Methods of reducing microbial resistance to drugs

ABSTRACT

The instant methods and compositions represent an advance in controlling drug resistance in microbes. AcrAB-like efflux pumps have been found to control resistance to drugs, even in highly resistant microbes. Accordingly, methods of treating infection, methods of screening for inhibitors of AcrAB-like efflux pumps, and methods of enhancing antimicrobial activity of drugs are provided. Pharmaceutical composition comprising an inhibitor of an AcrAB-like efflux pump and an antimicrobial agent are also provided.

GOVERNMENT FUNDING

This work was funded, at least in part, by a research grant from theU.S. Public Health Service GM 51661. The government, therefore, may havecertain rights in the invention.

BACKGROUND OF THE INVENTION

Different drugs used to inhibit microbial growth act by inhibitingdifferent targets. For example, the fluoroquinolone class of antibioticsact by inhibiting bacterial DNA synthesis. When used in treatment,fluoroquinolones are well absorbed orally, are found in respiratorysecretions in higher concentrations than in serum and are concentratedinside macrophages. In addition, fluoroquinolones are well tolerated andhave an excellent safety record in long-term therapy.

Antibiotic resistance, and in particular resistance to fluoroquinolones,has become a problem. Fluoroquinolone resistance in gram negativebacteria is principally caused by mutations affecting the targetproteins of the drugs. In the case of fluoroquinolones, these targetsare DNA gyrase and topoisomerase IV. In addition, mutations affectingregulatory genes such as marA, soxS or rob can cause fluoroquinoloneresistance (Oethinger et al. 1998. J. Antimicrob. Chemother. 41:111).Mar A is a transcriptional activator encoded by the marRAB operoninvolved in multiple antibiotic resistance (Alekshun et al. (1997)Antimicrob. Agents Chemother. 41, 2067–2075). The marRAB locus confersresistance to tetracycline, chloramphenicol, fluoroquinolones, nalidixicacid, rifampin, penicillin, as well as other compounds. However, marRABdoes not encode a multidrug efflux system. Rather, it controls theexpression of other loci important in directly mediating drugresistance, e.g., ompF, the gene for outer membrane porin, and the acrABgenes for the AcrAB efflux proteins.

AcrAB is a multidrug efflux pump (Nikaido, H. (1996) J. Bacteriol. 178,5853–5859; Okusu et al. (1996) J. Bacteriol. 178, 306–308) whose normalphysiological role is unknown, although it may assist in protection ofcells against bile salts in the mammalian small intestine (Thanassi etal. (1997) J. Bacteriol. 179, 2512–2518). The AcrAB operon isupregulated by MarA (Ma et al. (1995) Mol. Microbiol. 16, 45–55).Mutations in the repressor gene marR lead to overexpression of marA(Alekshun et al. (1997). Antimicrob. Agents Chemother. 41, 2067–2075;Cohen et al. (1993) J. Bacteriol. 175, 1484–492); Seoane et al. (1995)J. Bacteriol. 177, 3414–3419). The soxS gene encodes a MarA homolog(Alekshun et al. (1997) Antimicrob. Agents Chemother. 41, 2067–2075; Liet al. (1996) Mol. Microbiol. 20, 937–945; Miller et al. (1996) Mol.Microbiol. 21, 441–448) which also positively regulates acrAB (Ma et al.(1996) Mol. Microbiol. 19, 101–112).

The AcrAB pump primarily controls resistance to large, lipophilic agentsthat have difficulty penetrating porin channels, such as erythromycin,fusidic acid, dyes, and detergents, while leaving microbes susceptibleto small antibiotics that can diffuse through the channel, e.g.,tetracycline, chloramphenicol, and fluoroquinolones (Nikaido. 1996. J.Bacteriology 178:5853). Recently, the AcrAB pump has been found to beimportant in mediating resistance to other drugs used to controlmicrobial growth, e.g., non-antibiotic agents such as triclosan (FEMSMicrobiol Lett 1998 Sep. 15; 166: 305–9.

Microbes often become resistant to antibiotics and/or non-antibioticagents. This can occur by the acquisition of genes encoding enzymes thatinactivate the agents, modify the target of the agent, or result inactive efflux of the agent. Enzymes that inactivate syntheticantibiotics such as quinolones, sulfonamides, and trimethoprim have notbeen found. In the case of these antibiotics and natural products forwhich inactivating or modifying enzymes have not emerged, resistanceusually arises by target modifications (Spratt. 1994. Science 264:388).Improved methods for controlling drug resistance in microbes, inparticular in microbes that are highly resistant to drugs, would be oftremendous benefit.

SUMMARY OF THE INVENTION

The present invention is based, at least in part, on the discovery thatinactivation of the AcrAB locus makes even resistant microbial cellshypersusceptible to antibiotics and non-antibiotic drugs. Surprisingly,this is true even among highly resistant microbes which have chromosomalmutations that render them highly resistant to drugs.

Accordingly, in one aspect, the invention provides methods of treatingan infection caused by a drug resistant microbe in a subject byadministering a drug to which the microbe is resistant and an inhibitorof an AcrAB-like efflux pump to the subject such that the infection istreated.

In one embodiment, the drug is an antibiotic. In a preferred embodimentthe antibiotic is selected from the group consisting of afluoroquinolone and rifampin. In another embodiment, the drug is anon-antibiotic agent. In another embodiment, the drug is thenon-antibiotic agent, triclosan.

In one embodiment, the inhibitor of an AcrAB-like efflux pump isadministered prophylacticly. In another embodiment, the inhibitor of anAcrAB-like efflux pump is administered therapeutically.

In another aspect, the invention pertains to a method of treating afluoroquinolone resistant infection in a subject comprisingadministering a fluoroquinolone and an inhibitor of an AcrAB-like effluxpump to the subject to thereby treat a fluoroquinolone resistantinfection.

In another aspect, the invention pertains to a method of screening forcompounds which reduce drug resistance comprising: contacting a microbecomprising an AcrAB-like efflux pump with a test compound and aindicator compound and measuring the effect of the test compound onefflux of the indicator compound to thereby identify compounds whichreduce drug resistance by testing the ability of the test compound toinhibit the activity of an AcrAB efflux pump.

In one embodiment, the microbe is drug resistant. In a preferredembodiment, the microbial cell is highly drug resistant. In a morepreferred embodiment, the microbe is highly resistant tofluoroquinolones. In another embodiment, the microbial cell comprises atleast one mutation in a drug target gene. In another embodiment themicrobial cell cmprises at least two mutations in a drug target gene. Ina preferred embodiment, a mutation is present in a gene selected fromthe group consisting of: gyrase (gyrA), topoisomerase (parC), RNApolymerase, and fabI

In one embodiment, the subject assay includes detecting the ability ofthe compound to reduce fluoroquinolone resistance in a microbe.

In another aspect, the invention provides a method of screening forcompounds which specifically inhibit the activity of an AcrAB-likeefflux pump comprising:

i) contacting a microbe comprising an AcrAB-like efflux pump with a testcompound and an indicator compound;

ii) testing the ability of the compound to inhibit the activity of anAcrAB-like efflux pump;

iii) testing the ability of the compound to inhibit the activity of anon-AcrAB efflux pump;

iv) and identifying compounds which inhibit the activity of anAcrAB-like efflux pump and non a non-AcrAB-like efflux pump to therebyidentify compounds which specifically block an AcrAB-like efflux pump.

In yet another aspect, the invention provides a method of enhancing theantimicrobial activity of a drug comprising: contacting a microbe thatis highly resistant to one or more drugs with a drug to which themicrobe is resistant and an inhibitor of an AcrAB-like efflux pump tothereby enhance the antimicrobial activity of a drug.

In one embodiment, the step of contacting occurs ex vivo. In oneembodiment, the microbe is contacted with a non-antibiotic agent and aninhibitor of an AcrAB-like efflux pump. In one embodiment, thenon-antibiotic agent is selected from the group consisting of:cyclohexadine, quaternary ammonium compounds, pine oil, triclosan, andcompound generally regarded as safe (GRAS).

In another aspect, the invention provides a pharmaceutical compositioncomprising an inhibitor of an AcrAB-like efflux pump and an antibiotic.In one embodiment, the pharmaceutical composition further comprises apharmaceutically acceptable carrier. In a preferred embodiment, theantibiotic is selected from the group consisting of fluoroquinolone andrifampin.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 shows accumulation of ciprofloxacin (CIP) by energized cells.[¹⁴C-]ciprofloxacin uptake by fluoroquinolone-resistant mutants derivedin vitro from E. coli-K12 strains AG100 and AG112 was assayed at 30° C.at equilibrium after addition of 10 μM ciprofloxacin. Cells carriedeither the wild-type acrAB gene (solid bars) or an acrAB-deletion(hashed bars).

DETAILED DESCRIPTION OF THE INVENTION

The present invention represents an advance in controlling resistance todrugs which are substrates of AcrAB-like efflux pumps. In preferredembodiments, the methods of the invention can be used to controlresistance to antibiotic agents and non-antibiotic agents. In aparticularly preferred embodiment, drugs which are substrates ofAcrAB-like efflux pumps include antibiotics, e.g., fluoroquinolones orrifampin. The invention further pertains to AcrAB-like efflux pumpinhibitors obtained using the instant methods and their methods of use.In another particularly preferred embodiment, drugs includenon-antibiotic agents, e.g., triclosan. The subject methods areeffective in controlling drug resistance even among highly resistantmicrobes that bear chromosomal mutations which alter drug targetmolecules. For example, analysis of microbial mutants has revealed thatmutations in the fluoroquinolone target gene gyrA, the regulatory genemarR, and additional, as yet unidentified genes probably affectingAcrAB-mediated efflux of ciprofloxacin all contributed tofluoroquinolone resistance. Surprisingly, inactivation of the AcrABlocus made even highly resistant cells hypersusceptible tofluoroquinolones and certain other unrelated drugs even amongtopoisomerase mutants. These studies indicate that blocking the functionof AcrAB-like efflux pumps reduces even high-level drug resistance.

Accordingly, the invention provides, inter alia, methods of screeningfor compounds which reduce drug resistance, in particular to antibioticsand non-antibiotic agents such as fluoroquinolones and triclosan, andmethods of screening for compounds that specifically block an AcrAB-likeefflux pump. In addition, the invention provides methods of enhancingthe antimicrobial activity of a drug and methods of treating infection.

Before further description of the invention, certain terms employed inthe specification, examples and appended claims are, for convenience,collected here.

I. Definitions

As used herein the term “infection” includes the presence of a microbein or on a subject which, if its growth were inhibited, would result ina benefit to the subject. As such, the term “infection” in addition toreferring to the presence of pathogens also includes normal flora whichis not desirable, e.g., on the skin of a burn patient or in thegastrointestinal tract of an immunocompromised patient. As used herein,the term “treating” refers to the administration of a compound to asubject, for prophylactic and/or therapeutic purposes. The term“administration” includes delivery to a subject, e.g., by anyappropriate method which serves to deliver the drug to the site of theinfection. Administration of the drug can be, e.g., oral, intravenous,or topical.

As used herein, the term “drug” includes compounds which are substratesof AcrAB-like efflux pumps. The term “drug” includes compounds whichreduce the growth of a microbe e.g., which reduce the ability of amicrobe to produce infection in a host, or which reduce the ability of amicrobe to multiply or remain infective in the environment. Such drugsinclude antibiotic agents and non-antibiotic agents. The term “drug”includes antiinfective compounds which are static or cidal for microbes,e.g., an antimicrobial compound which inhibits the growth and/orviability of a microbe. Preferred antiinfective compounds increase thesusceptibility of microbes to antibiotics or decrease the infectivity orvirulence of a microbe. Substrates of the AcrAB-like efflux pumps can bereadily identified using methods known in the art and described infurther detail herein. For example, putative drugs which are substratesof AcrAB-like efflux pumps can be labeled, e.g., radioactively, andtheir export from a microbial cell tested in microbial cells whichpossess AcrAB-like efflux pumps and in microbial cells which lackAcrAB-like efflux pumps. Those agents which are present at a higherintracellular concentration in microbes that lack such pumps than inmicrobes that possess such pumps are drugs which are substrates of thepump.

The term “drug” includes the antimicrobial agents to which the Marphenotype has been shown to mediate resistance and, as such, includesdisinfectants, antiseptics, and surface delivered compounds. Forexample, antibiotics, biocides, or other type of antibacterialcompounds, including agents which induce oxidative stress agents, andorganic solvents are included in this term. The term “drug” alsoincludes biocidal agents. The term “biocidal” is art recognized andincludes an agent that those ordinarily skilled in the art prior to thepresent invention believed would kill a cell “non-specifically,” or abroad spectrum agent whose mechanism of action is unknown, e.g., priorto the present invention, one of ordinary skill in the art would nothave expected the agent to be target-specific. Examples of biocidalagents include paraben, chlorbutanol, phenol, alkylating agents such asethylene oxide and formaldehyde, halides, mercurials and other heavymetals, detergents, acids, alkalis, and chlorhexidine. Other biocidalagents include: triclosan, pine oil, quaternary amine compounds such asalkyl dimethyl benzyl ammonium chloride, chloroxylol, chlorhexidine,cyclohexidine, triclocarbon, and disinfectants. The term “bactericidal”refers to an agent that can kill a bacterium; “bacteriostatic” refers toan agent that inhibits the growth of a bacterium.

The term “antibiotic” is art recognized and includes antimicrobialagents synthesized by an organism in nature and isolated from thisnatural source, and chemically synthesized drugs. The term includes butis not limited to: polyether ionophore such as monensin and nigericin;macrolide antibiotics such as erythromycin and tylosin; aminoglycosideantibiotics such as streptomycin and kanamycin; β-lactam antibioticssuch as penicillin and cephalosporin; and polypeptide antibiotics suchas subtilisin and neosporin. Semi-synthetic derivatives of antibiotics,and antibiotics produced by chemical methods are also encompassed bythis term. Chemically-derived antimicrobial agents such as isoniazid,trimethoprim, quinolones, fluoroquinolones and sulfa drugs areconsidered antibacterial drugs, and the term antibiotic includes these.It is within the scope of the screens of the present invention toinclude compounds derived from natural products and compounds that arechemically synthesized.

In contrast to the term “biocidal,” an antibiotic or an “anti-microbialdrug approved for human use” is considered to have a specific moleculartarget in a microbial cell. Preferably a microbial target of atherapeutic agent is sufficiently different from its physiologicalcounterpart in a subject in need of treatment that the antibiotic ordrug has minimal adverse effects on the subject.

The phrase “non-antibiotic agent” includes substrates of an acrAB-likeefflux pump which are not art recognized as being antibiotics. Exemplarynon-antibiotic agents include, e.g., biocides, disinfectants orantiinfectives. Non antibiotic agents also include substrates of anacrAB-like efflux pump which are incorporated into consumer goods, e.g.,for topical use on a subject or as cleaning products.

As used herein, the term “fluoroquinolone” includes quinolonessubstituted with at least one fluorine atom. Preferred fluoroquinolonesinclude compounds with the carbonyl at the 4 position. Preferredpositions for fluorine substitution include the 5, 6, and 7 positions.Derivatives include compounds with additional substituents such as,although not limited to, NR′R″, CN, NO₂, F, Cl, Br, I, CF₃, CCl₃, CHF₂,CHCl₂, CONR′R″, S(O)NR′R″, CHO, OCF₃, OCCl₃, SCF₃, SCCl₃, COR′, CO₂R′,and OR′ and wherein R′ and R″ are each independently hydrogen, C₁–C₄alkyl, C₂–C₄ alkenyl, C₂–C₄ alkynyl or optionally substituted cyclic orheterocyclic groups. The term fluoroquinolone also encompasses compoundswith heterocyclic substitutions at the seven position, such asnaphthyridinones. Preferred substituents include piperazinyl groups andother heterocyclic groups, carboxylic acid groups and substituted orunsubstituted alkyl groups. One example of a preferred fluoroquinoloneis shown below, wherein X is CH or N or CR₆, and where R₁ is lower alkylincluding cycloalkyl groups, or optionally substituted with halogens;R₂, R₃, and R₄ are each independently substituted or unsubstituted loweralkyl or hydrogen; R₅ is fluorine, hydrogen or amino; and R₆ is hydrogenor fluorine.

As used herein, the term “multiple drug resistance (MDR)” includesresistance to both antibiotic and non-antibiotic compounds. MDR resultsfrom the increased transcription of a chromosomal or plasmid encodedgenetic locus in an organism, e.g., a marRAB locus, that results in theability of the organism to minimize the toxic effects of a compound towhich it has been exposed, as well as to other non-related compounds,e.g., by stimulating an efflux pump(s) or microbiological catabolic ormetabolic processes. As used herein, the phrase “microbes which areresistant to drugs or drug resistant microbes” includes microbes thatare characterized by a mutation in a target gene or by increasedtranscription of a genetic locus that affects drug resistance, e.g., anefflux pump gene.

As used herein, the phrase “microbes which are resistant to drugs ordrug resistant microbes” includes microbes that are characterized bymutations in a gene that is the target of a drug.

As used herein, the phrase “microbes which are highly resistant to drugsor highly drug resistant microbes” includes microbes that arecharacterized by mutations in multiple (i.e., more than one) gene thataffects drug resistance. Preferably, a microbe that is highly resistantto drugs is characterized by at least two of the following three traits:(1) it comprises at least one mutation in a gene encoding a drug targetthat renders the microbe resistant to one or more drugs (e.g., a gyrase,fabI or topoisomerase mutation); (2) it comprises a second mutation (tothe same gene or a different gene than in (1)) that increases drugresistance; and (3) it has increased expression of at least one effluxpump (e.g., as a result of increased transcription of the mar locus).The term “mutation” includes an alteration (e.g., a substitution,deletion, or insertion) of at least one nucleotide in the sequence of anucleic acid molecule (either chromosomal or episomal) in a microbewhich is capable of influencing drug resistance. Such a mutation canresult, e.g., in altered gene regulation in the microbe or in theexpression of an altered polypeptide. Preferably, such mutations are ingenes which encode the target of the drug to which the microbe isresistant.

In one embodiment, the drug is an antibiotic. In a preferred embodiment,the drug is a fluoroquinolone.

In another embodiment, the drug is a non-antibiotic agent, e.g.,triclosan, pine oil, quaternary amine compounds such as alkyl dimethylbenzyl ammonium chloride, chloroxylol, triclocarbon, or a disinfectant,described in further detail herein. In a particularly preferredembodiment, the drug is a substrate of an acrAB-like efflux pump whichis not a non-antibiotic agent.

Microbes that are highly resistant to drugs are more resistant to drugsthan microbes that that are characterized by only one of the precedingtraits. In general, antibiotics, when tested for their effect on thegrowth of such highly resistant microbes, will yield a minimalinhibitory concentration (MIC) from between about 2-fold to more than100-fold higher than that observed for a microbe that is characterizedby only one of the above traits or a microbe that is multiply antibioticresistant, but not highly resistant to drugs.

As used herein the term “drug target” includes molecules which are actedon by drugs and which, in a non-resistant microbe, are altered such thatthey do not retain their normal function and the growth of the microbeis inhibited. For example, exemplary drug targets include: the DNAgyrase and topoisomerase molecules, which are targets of fluoroquinoloneantibiotics; RNA polymerase, which is a target of rifampin; and FabI,which is a target of triclosan (Heath R J, e.t. al. 1999. J Biol Chem;274; Levy C W et al. 1999. Nature 398: 383–4; McMurry L M; et al. 1998.Nature. 394: 531). In one embodiment, a drug target is not FabI.

As used herein the term “AcrAB-like efflux pump” includes efflux pumpsthat have homology with the AcrAB efflux pump of E. coli. The AcrAB pumpis a resistance, nodulation, and division (RND)-type pump. RND pumpshave 12 transmembrane helices. The acrA and acrB genes have been clonedand sequenced (Ma et al. 1993. J. Bacteriol. 175:6229). The sequences ofAcrAB in E. coli are deposited as GenBank accession number U00734. TheAcrAB genes have other homologs in E. coli, as well as homologs in otherspecies of bacteria. For example, homologs of the AcrAB efflux pump havebeen identified in Haemophilus influenzae, (Sanchez et al. 1997. J.Bacteriol. 179:6855) and in Salmonella typhimurium (Nikaido et al. 1998.J. Bacteriol. 180:4686). Exemplary homologues of AcrAB include: MtrCD,MexAB-OprM, MexCD-OprJ, MexEF-OprN, and YhiUV. Such homologs can bereadily identified by one of ordinary skill in the art based on sharedhomology and structure with the AcrAB pump and/or based on similaritiesin the compounds which they export. Isolation of novel AcrAB-like effluxpumps from other microbes can be carried out using techniques which areknown in the art, e.g., nucleic acid hybridization and functionalcloning.

As used herein the term “AcrAB-like efflux pump inhibitor” refers to acompound which interferes with the ability of an AcrAB-like efflux pumpto export a compound which it is normally capable of exporting in theabsence of such an inhibitor. Such inhibitors can inhibit the activityof an AcrAB-like efflux pump directly, e.g., by blocking the pump, orindirectly, e.g., by reducing transcription of acrA-like and/oracrB-like genes. Inhibitors of AcrAB-like efflux pumps can inhibit thegrowth of resistant and/or highly resistant microbes which used alone,or they may potentiate the activity of a drug to which the microbe isresistant.

As used herein the term “non-AcrAB-like efflux pump” includes effluxpumps which are not related to the AcrAB efflux pump of E. coli. Suchpumps include, e.g., major facilitator pumps, membrane fusion proteins,and ABC (ATP-binding cassette) pumps.

As used herein the term “growth” in reference to the growth of a microbeincludes the reproduction or population expansion of the microbe, e.g.,increase in numbers rather than increase in size. The term also includesmaintenance of on-going metabolic processes of a microbe, e.g., thoseprocesses that keep the cell alive when the cell is not dividing.

As used herein the term “reporter gene” includes any gene which encodesan easily detectable product which gene is operably linked to apromoter. By operably linked it is meant that under appropriateconditions an RNA polymerase may bind to the promoter of the regulatoryregion and proceed to transcribe the nucleotide sequence of the reportergene. In preferred embodiments, a reporter gene construct consists of apromoter linked to a reporter gene. In certain embodiments, however, itmay be desirable to include other sequences, e.g., transcriptionalregulatory sequences, in the reporter gene construct. For example,modulation of the activity of the promoter may be affected by alteringthe RNA polymerase binding to the promoter region, or, alternatively, byinterfering with initiation of transcription or elongation of the mRNA.Thus, sequences which are herein collectively referred to astranscriptional regulatory elements or sequences may also be included inthe reporter gene construct. In addition, the construct may includesequences of nucleotides that alter translation of the resulting mRNA,thereby altering the amount of reporter gene product.

As used herein the term “test compound” includes reagents tested usingthe assays of the invention to determine whether they modulate anAcrAB-like efflux pump activity. More than one test compound, e.g., aplurality of test compounds, can be tested at the same time for theirability to modulate the activity of an AcrAB-like efflux pump in ascreening assay.

Compounds that can be tested in the subject assays include antibioticand non-antibiotic compounds. Exemplary test compounds which can bescreened for activity include, but are not limited to, peptides,non-peptidic compounds, nucleic acids, carbohydrates, small organicmolecules (e.g., polyketides), and natural product extract libraries.The term “non-peptidic compound” is intended to encompass compounds thatare comprised, at least in part, of molecular structures different fromnaturally-occurring L-amino acid residues linked by natural peptidebonds. However, “non-peptidic compounds” are intended to includecompounds composed, in whole or in part, of peptidomimetic structures,such as D-amino acids, non-naturally-occurring L-amino acids, modifiedpeptide backbones and the like, as well as compounds that are composed,in whole or in part, of molecular structures unrelated tonaturally-occurring L-amino acid residues linked by natural peptidebonds. “Non-peptidic compounds” also are intended to include naturalproducts.

As used herein the phrase “indicator compound” includes compounds whichare normally exported by an AcrAB-like efflux pump. Indicator compoundsare used as markers of AcrAB-like efflux pump activity in order todetermine the effect of a test compound on the activity of an AcrAB-likeefflux pump. Exemplary indicator compounds include, e.g., antibioticsand dyes.

II. Inhibitors of AcrAB-Like Efflux Pumps

Known inhibitors of efflux pumps can be used in the methods andcompositions of the invention. Exemplary inhibitors have been describedpreviously in PCT published patent application WO96/33285 (includingL-phenylalanyl-L-arginyl-β-naphthylamide). Methods for testing compoundsfor efflux pump inhibition are also described therein. Other usefulinhibitors include ethanol (concentrations of about 4%), methanol,hexane and minocycline. Still other inhibitors include antisense nucleicacids and ribozymes directed against the gene(s) encoding the effluxpump. Characteristics of other efflux pump inhibitors are described,e.g., in WO 96/33285.

Other exemplary AcrAB-like efflux pump inhibitors include, e.g.,antisense nucleic acids which bind to AcrAB genes and preventtranscription or translation thereof. Antibodies which bind efflux pumpsor proteins which regulate the expression of efflux pumps are anotherclass of inhibitors. Still other inhibitors include genes which repressexpression of the efflux pumps or regulatory loci (such as marR) whichregulate expression of efflux pumps. Increasing the amount of such genesor the expression products thereof can reduce the expression of effluxpumps in microbes.

As mentioned above, the invention embraces antisense nucleic acids,including oligonucleotides, that selectively bind to a nucleic acidmolecule encoding an efflux pump (e.g. acrA) or a molecule whichregulates expression of an efflux pump (e.g. marA, rob or soxS). As usedherein, the term “antisense oligonucleotide” or “antisense molecule”describes an oligonucleotide that is an oligoribonucleotide,oligodeoxyribonucleotide, modified oligoribonucleotide, or modifiedoligodeoxyribonucleotide which hybridizes under physiological conditionsto DNA comprising a particular gene or to an RNA transcript of that geneand, thereby, inhibits the transcription of that gene and/or thetranslation of that RNA. The antisense molecules are designed so as tointerfere with transcription or translation of a target gene uponhybridization with the target gene or transcript. Those skilled in theart will recognize that the exact length of the antisenseoligonucleotide and its degree of complementarity with its target willdepend upon the specific target selected, including the sequence of thetarget and the particular bases which comprise that sequence. It ispreferred that the antisense oligonucleotide be constructed and arrangedso as to bind selectively with the target under physiologicalconditions, i.e., to hybridize substantially more strongly to the targetsequence than to any other sequence in the target cell underphysiological conditions. Based upon the nucleic acid sequence of a geneof interest, one of skill in the art can easily choose and synthesizeany of a number of appropriate antisense molecules for use in accordancewith the present invention. In order to be sufficiently selective andpotent for inhibition, such antisense oligonucleotides should compriseat least 10 and, more preferably, at least 15 consecutive bases whichare complementary to the target, although in certain cases modifiedoligonucleotides as short as 7 bases in length have been usedsuccessfully as antisense oligonucleotides (Wagner et al., NatureBiotechnol. 14:840–844, 1996). Most preferably, the antisenseoligonucleotides comprise a complementary sequence of 20–30 bases.Although oligonucleotides may be chosen which are antisense to anyregion of the gene or RNA transcripts, in preferred embodiments theantisense oligonucleotides correspond to N-terminal or 5′ upstream sitessuch as translation initiation, transcription initiation or promotersites. In addition, 3′-untranslated regions may be targeted. Inaddition, the antisense is targeted, preferably, to sites in which RNAsecondary structure is not expected and at which proteins are notexpected to bind.

In embodiment, the antisense oligonucleotides of the invention may becomposed of “natural,” i.e., unmodified deoxyribonucleotides,ribonucleotides, or any combination thereof. That is, the 5′ end of onenative nucleotide and the 3′ end of another native nucleotide may becovalently linked, as in natural systems, via a phosphodiesterinternucleoside linkage. These oligonucleotides may be prepared bystandard methods which may be carried out manually or by an automatedsynthesizer. They also may be produced recombinantly by vectors.

In preferred embodiments, however, the antisense oligonucleotides of theinvention also may include “modified” oligonucleotides. That is, theoligonucleotides may be modified in a number of ways as compared tonaturally occurring oligonucleotides which do not prevent them fromhybridizing to their target but which enhance their stability ortargeting or which otherwise enhance their therapeutic effectiveness.

The term “modified oligonucleotide” as used herein describes anoligonucleotide in which (1) at least two of its nucleotides arecovalently linked via a synthetic internucleoside linkage (i.e., alinkage other than a phosphodiester linkage between the 5′ end of onenucleotide and the 3′ end of another nucleotide) and/or (2) a chemicalgroup not normally associated with nucleic acids has been covalentlyattached to the oligonucleotide. Preferred synthetic internucleosidelinkages are phosphorothioates, alkylphosphonates, phosphorodithioates,phosphate esters, alkylphosphonothioates, phosphoramidates, carbamates,carbonates, phosphate triesters, acetamidates, carboxymethyl esters andpeptides.

The term “modified oligonucleotide” also encompasses oligonucleotideswith a covalently modified base and/or sugar. For example, modifiedoligonucleotides include oligonucleotides having backbone sugars whichare covalently attached to low molecular weight organic groups otherthan a hydroxyl group at the 3′ position and other than a phosphategroup at the 5′ position. Thus modified oligonucleotides may include a2′-O-alkylated ribose group. In addition, modified oligonucleotides mayinclude sugars such as arabinose instead of ribose. The presentinvention, thus, provides preparations containing modified antisensemolecules that are complementary to and can hybridize with, underphysiological conditions, nucleic acids encoding mar/sox/rob or effluxpump polypeptides, together with one or more carriers.

As described above, the invention further embraces the use of antibodiesor fragments of antibodies having the ability to selectively bind toefflux pumps, as well as polypeptides which regulate the expression ofefflux pumps. The term “antibody” includes polyclonal and monoclonalantibodies, or fragments thereof, prepared according to conventionalmethodology.

Accordingly, in another embodiment, antibodies to AcrAB-like effluxpumps can be used as efflux pump inhibitors. Polyclonal anti-efflux pumpantibodies can be prepared as described above by immunizing a suitablesubject with an immunogen derived from an AcrAB-like efflux pump. Theanti-efflux pump antibody titer in the immunized subject can bemonitored over time by standard techniques, such as with an enzymelinked immunosorbent assay (ELISA) using immobilized efflux pump. Ifdesired, the antibody molecules directed against efflux pump can beisolated from the mammal (e.g., from the blood) and further purified bywell known techniques, such as protein A chromatography to obtain theIgG fraction. At an appropriate time after immunization, e.g., when theanti-efflux pump antibody titers are highest, antibody-producing cellscan be obtained from the subject and used to prepare monoclonalantibodies by standard techniques, such as the hybridoma techniqueoriginally described by Kohler and Milstein (1975, Nature 256:495–497)(see also, Brown et al. (1981) J. Immunol 127:539–46; Brown et al.(1980) J. Biol Chem 255:4980–83; Yeh et al (1976) PNAS 76:2927–31; andYeh et al. (1982) Int. J. Cancer 29:269–75), the more recent human Bcell hybridoma technique (Kozbor et al. (1983) Immunol Today 4:72), theEBV-hybridoma technique (Cole et al. (1985), Monoclonal Antibodies andCancer Therapy, Alan R. Liss, Inc., pp. 77–96) or trioma techniques. Thetechnology for producing monoclonal antibody hybridomas is well known(see generally R. H. Kenneth, in Monoclonal Antibodies: A New DimensionIn Biological Analyses, Plenum Publishing Corp., New York, N.Y. (1980);E. A. Lerner (1981) Yale J. Biol. Med., 54:387–402; M. L. Gefter et al.(1977) Somatic Cell Genet., 3:231–36). Briefly, an immortal cell line(typically a myeloma) is fused to lymphocytes (typically splenocytes)from a mammal immunized with a efflux pump immunogen as described above,and the culture supernatants of the resulting hybridoma cells arescreened to identify a hybridoma producing a monoclonal antibody thatbinds specifically to an AcrAB-like efflux pump.

Any of the many well known protocols used for fusing lymphocytes andimmortalized cell lines can be applied for the purpose of generating ananti-efflux pump monoclonal antibody (see, e.g., G. Galfre et al. (1977)Nature 266:55052; Gefter et al. Somatic Cell Genet., cited supra;Lerner, Yale J. Biol. Med., cited supra; Kenneth, Monoclonal Antibodies,cited supra). Moreover, the ordinary skilled worker will appreciate thatthere are many variations of such methods which also would be useful.Typically, the immortal cell line (e.g., a myeloma cell line) is derivedfrom the same mammalian species as the lymphocytes. For example, murinehybridomas can be made by fusing lymphocytes from a mouse immunized withan immunogenic preparation of the present invention with an immortalizedmouse cell line. Preferred immortal cell lines are mouse myeloma celllines that are sensitive to culture medium containing hypoxanthine,aminopterin and thymidine (“HAT medium”). Any of a number of myelomacell lines may be used as a fusion partner according to standardtechniques, e.g., the P3-NS1/1-Ag4-1, P3-x63-Ag8.653 or Sp2/O-Ag14myeloma lines. These myeloma lines are available from the American TypeCulture Collection (ATCC), Rockville, Md. Typically, HAT-sensitive mousemyeloma cells are fused to mouse splenocytes using polyethylene glycol(“PEG”). Hybridoma cells resulting from the fusion are then selectedusing HAT medium, which kills unfused and unproductively fused myelomacells (unfused splenocytes die after several days because they are nottransformed). Hybridoma cells producing a monoclonal antibody of theinvention are detected by screening the hybridoma culture supernatantsfor antibodies that bind efflux pump, e.g., using a standard ELISAassay.

As an alternative to preparing monoclonal antibody-secreting hybridomas,a monoclonal anti-efflux pump antibody can be identified and isolated byscreening a recombinant combinatorial immunoglobulin library (e.g., anantibody phage display library) with efflux pump to thereby isolateimmunoglobulin library members that bind efflux pumps. Kits forgenerating and screening phage display libraries are commerciallyavailable (e.g., the Pharmacia Recombinant Phage Antibody System,Catalog No. 27-9400-01; and the Stratagene SurfZAP™ Phage Display Kit,Catalog No. 240612). Additionally, examples of methods and reagentsparticularly amenable for use in generating and screening antibodydisplay library can be found in, for example, Ladner et al. U.S. Pat.No. 5,223,409; Kang et al. International Publication No. WO 92/18619;Dower et al. International Publication No. WO 91/17271; Winter et al.International Publication WO 92/20791; Markland et al. InternationalPublication No. WO 92/15679; Breitling et al. International PublicationWO 93/01288; McCafferty et al. International Publication No. WO92/01047; Garrard et al. International Publication No. WO 92/09690;Ladner et al. International Publication No. WO 90/02809; Fuchs et al.(1991) Bio/Technology 9:1370–1372; Hay et al. (1992) Hum AntibodHybridomas 3:81–85; Huse et al. (1989) Science 246:1275–1281; Griffithset al. (1993) EMBO J. 12:725–734; Hawkins et al. (1992) J Mol Biol226:889–896; Clarkson et al. (1991) Nature 352:624–628; Gram et al.(1992) PNAS 89:3576–3580; Garrad et al. (1991) Bio/Technology9:1373–1377; Hoogenboom et al. (1991) Nuc Acid Res 19:4133–4137; Barbaset al. (1991) PNAS 88:7978–7982; and McCafferty et al. Nature (1990)348:552–554.

Additionally, recombinant anti-efflux pump antibodies, such as chimericand humanized monoclonal antibodies, comprising both human and non-humanportions, which can be made using standard recombinant DNA techniques,are within the scope of the invention. Such chimeric and humanizedmonoclonal antibodies can be produced by recombinant DNA techniquesknown in the art, for example using methods described in Robinson et alInternational Patent Publication PCT/US86/02269; Akira, et al. EuropeanPatent Application 184,187; Taniguchi, M., European Patent Application171,496; Morrison et al. European Patent Application 173,494; Neubergeret al. PCT Application WO 86/01533; Cabilly et al. U.S. Pat. No.4,816,567; Cabilly et al. European Patent Application 125,023; Better etal. (1988) Science 240:1041–1043; Liu et al. (1987) PNAS 84:3439–3443;Liu et al. (1987) J. Immunol. 139:3521–3526; Sun et al. (1987) PNAS84:214–218; Nishimura et al. (1987) Canc. Res. 47:999–1005; Wood et al.(1985) Nature 314:446–449; and Shaw et al. (1988) J. Natl Cancer Inst.80:1553–1559); Morrison, S. L. (1985) Science 229:1202–1207; Oi et al.(1986) BioTechniques 4:214; Winter U.S. Pat. No. 5,225,539; Jones et al.(1986) Nature 321:552–525; Verhoeyan et al. (1988) Science 239:1534; andBeidler et al. (1988) J. Immunol. 141:4053–4060.

III. Methods of Screening for Novel Inhibitors

In one aspect, the invention provides a method for screening for aninhibitor of an AcrAB-like efflux pump. In this method, microbesexpressing an AcrAB-like efflux pump are contacted with a test compoundand a indicator compound. The test compound is a compound to be testedfor its ability to inhibit an AcrAB-like efflux pump. An indicatorcompound is one which is normally exported by the AcrAB-like effluxpump. Using the subject methods the ability of a test compound toinhibit the activity of an AcrAB-like efflux pump is demonstrated bydetermining whether the intracellular concentration of the indicatorcompound (e.g., a fluoroquinolone or a dye) is elevated in the presenceof the test compound. If the intracellular concentration of theindicator compound is increased in the presence of the test compound ascompared to the intracellular concentration in the absence of the testcompound, then the test compound can be identified as an inhibitor of anAcrAB-like efflux pump. Thus, one can determine whether or not the testcompound is an inhibitor of an AcrAB-like efflux pump by showing thatthe test compound affects the ability of an AcrAB-like efflux pumppresent in the microbe to export the indicator compound.

The “intracellular concentration” of an indicator compound includes theconcentration of the indicator compound inside the outermost membrane ofthe microbe. The outermost membrane of the microbe can be, e.g., acytoplasmic membrane. In the case of Gram-negative bacteria, therelevant “intracellular concentration” is the concentration in thecellular space in which the indicator compound localizes, e.g., thecellular space which contains a target of the indicator compound.

Inhibitors identified using the subject methods can act directly on anAcrAB-like pump, e.g., by steric inhibition, or can act indirectly,e.g., influencing a more distal event, e.g., by modulating transcriptionof genes involved in the expression of the pump.

In one embodiment, the method comprises detecting the ability of thecompound to reduce fluoroquinolone resistance in a microbe. For example,in one embodiment, the indicator compound comprises a fluoroquinoloneand the effect of the test compound on the intracellular concentrationof fluoroquinolone in the microbe is measured. In one embodiment, anincrease in the intracellular concentration of fluoroquinolone can bemeasured directly, e.g., in an extract of microbial cells. For example,accumulation of a radiolabelled fluoroquinolone, e.g.,[¹⁴C-]ciprofloxacin can be determined using standard techniques. Forinstance, microbes can be contacted with a radiolabelled fluoroquinoloneas an indicator composition in the presence and absence of a testcompound. The concentration of the fluoroquinolone inside the cells canbe measured at equilibrium by harvesting cells from the two groups (withand without test compound) and cell associated radioactivity measuredwith a liquid scintillation counter. In another embodiment, an increasein the intracellular concentration of fluoroquinolone can be measuredindirectly, e.g., by a showing that a given concentration offluoroquinolone when contacted with the microbe is sufficient to inhibitthe growth of the microbe in the presence of the test compound, but notin the absence of the test compound.

In one embodiment of the subject assays, the step of determining whetherthe intracellular concentration of the indicator compound is elevated isaccomplished by measuring a decrease in the minimal inhibitoryconcentration (MIC) of the indicator compound. Such an assay can beperformed using a standard methods, e.g., an antibiotic disc assay.

In another embodiment, measurement of the intracellular concentration ofan indicator compound can be facilitated by using an indicator compoundwhich is readily detectable by spectroscopic means. Such a compound maybe, for example, a dye, e.g., a basic dye, or a fluorophore. Exemplaryindicator compounds include: acridine, ethidium bromode, gentian violet,malachite green, methylene blue, beenzyn viologen, bromothymol blue,toluidine blue, methylene blue, rose bengal, alcyan blue, ruthenium red,fast green, aniline blue, xylene cyanol, bromophenol blue, coomassieblue, bormocresol purple, bromocresol green, trypan blue, and phenolred.

In such an assay, the effect of the test compound on the ability of thecell to export the indicator compound can be measured spectroscopically.For example, the intracellular concentration of the dye or fluorophorecan be determined indirectly, by determining the concentration of theindicator compound in the suspension medium or by determining theconcentration of the indicator compound in the cells. This can be done,e.g., by extracting the indicator compound from the cells or by visualinspection of the cells themselves.

In another embodiment, the presence of an indicator compound in amicrobe can be detected using a reporter gene which is sensitive to thepresence of the indicator compound. Exemplary reporter genes are knownin the art. For example, a reporter gene can provide a colorometric readout or an enzymatic read out of the presence of an indicator compound.In yet another embodiment, a reporter gene whose expression is inducibleby the presence of a drug in a microbe can be used. For example, amicrobe can be grown in the presence of a drug with and without aputative AcrAB-like efflux pump inhibitor. In cells in which the effluxpump is inhibited, the concentration of the drug will be increased andthe reporter gene construct will be expressed. By this method,AcrAB-like efflux pump inhibitors are identified by their ability toinhibit the export rate of the drug and, thus, to induce reporter geneexpression.

In another embodiment, a primary screening assay is used in which anindicator compound which does not comprise the drug of interest, e.g., afluoroquinolone or triclosan is employed. In one embodiment, upon theidentification of a test compound that increases the intracellularconcentration of the test compound, a secondary screening assay isperformed in which the effect of the same test compound on resistance tothe drug of interest, e.g., fluoroquinolone resistance, is measured.

In another aspect, the invention provides a method of screening forcompounds which specifically block an AcrAB-like efflux pump. In oneembodiment the method involves contacting a microbe comprising anAcrAB-like efflux pump with a test compound and a indicator compound.The test compound is then tested for its ability to block an AcrAB-likeefflux pump as described supra. The specificity of compounds which areidentified as candidate AcrAB inhibitory agents can then be tested fortheir ability to block a non-AcrAB efflux pump. Compounds which block anAcrAB-like efflux pump and not a non-AcrAB efflux pump can be identifiedas compounds that specifically block an AcrAB-like efflux pump.

In one embodiment, the use of a compound which is identified as aninhibitor of an AcrAB-like efflux pump in the subject methods preventsthe development of a drug resistant microbe or of a highly drugresistant microbe from a drug resistant microbe.

IV. Methods of Enhancing the Antimicrobial Activity of a Drug

In one aspect the invention pertains to methods of enhancing theantimicrobial activity of a drug by contacting a microbe that isresistant to one or more drugs with a drug to which the microbe isresistant and an inhibitor of an AcrAB-like efflux pump. In oneembodiment, the microbe is contacted with the drug and the inhibitor ofthe AcrAB-like efflux pump ex vivo. This method can be used, e.g., indisinfecting surfaces to prevent the spread of infection or in cleaningsurfaces which are fouled by microbial growth. Preferably, the drug usedto contact the microbe is a non-antibiotic drug. In a preferredembodiment, the microbe is contacted with triclosan and an inhibitor ofan AcrAB-like efflux pump.

In one embodiment, an AcrAB-like efflux pump inhibitor and a drug can becombined in a disinfectant, e.g., a cleaning product or a householdproduct for contacting with resistant microbes. Exemplary cleaningproducts can be used topically on a subject (e.g., as soaps or lotions)or can be used for cleaning surfaces. In one embodiment, an AcrAB-likeefflux pump inhibitor is itself a disinfectant.

V. Methods of Treating Microbial Infections

In one aspect the invention provides a method of treating a drugresistant infection in a subject comprising administering a drug towhich a microbe is resistant and an inhibitor of an AcrAB-like effluxpump to the subject. As used herein the term “administration” includescontacting a drug with a subject, e.g. in vivo and/or in vitro. Thus, anefflux pump inhibitor and a drug can be administered for in vivotreatment or can be used topically, e.g., on skin or the eyes.

In one embodiment, the drug is an antibiotic, e.g., a fluoroquinolone.In another embodiment, the drug is a non-antibiotic composition, e.g.,triclosan. In another embodiment the infection to be treated is onenormally treated with a non-antibiotic composition. In anotherembodiment, the infection to be treated is not one normally treated witha non-antibiotic composition.

In one embodiment of the treatment method, the efflux pump inhibitor andthe drug are administered separately to the subject. In anotherembodiment, the efflux pump inhibitor and the drug are administeredsimultaneously. In one embodiment, the simultaneous administration ofthe drug and the AcrAB-like efflux pump inhibitor is facilitated by theadministration of a pharmaceutical composition comprising both an effluxpump inhibitor and a drug to which the microbe is resistant.

The amount of efflux pump inhibitor to be administered to a subject is atherapeutically effective amount, e.g., for an efflux pump inhibitor, anamount sufficient to reduce efflux pump activity. The dosage of effluxpump inhibitor to be administered to a subject that would benefit fromtreatment with a drug, e.g. a patient having an infection with amicrobe, can readily be determined by one of ordinary skill in the art.Ideally, the dosage of efflux pump inhibitor administered will besufficient to reduce efflux pump activity such that standard doses ofdrugs have a therapeutic effect, e.g., result in a benefit to thesubject, e.g., by inhibiting microbial growth. The phrase “therapeuticeffect” refers to an amelioration of symptoms or a prolongation ofsurvival in a subject. In a preferred embodiment, a therapeutic effectis an elimination of a microbial infection.

In one embodiment, the subject is an avian subject, e.g., a chicken or aturkey. In another embodiment, the subject is a mammalian subject, e.g.,a horse, sheep, pig, cow, dog, or cat. In a preferred embodiment, thesubject is a human subject.

In one embodiment, an infection in a subject is treated prophylacticly.The term “prophylactic” treatment refers to treating a subject who isnot yet infected, but who is susceptible to, or at risk of an infection.In one embodiment, the efflux pump inhibitor is administered prior toexposure to an infectious agent. In another embodiment, an efflux pumpinhibitor is administered to a subject prior to the exposure of thesubject to a drug resistant organism. The term “therapeutic” treatmentrefers to administering a compound to a subject already suffering froman infection.

In one embodiment an efflux pump inhibitor and/or drug may beadministered in prodrug form, e.g., may be administered in a form whichis modified within the cell to produce the functional form of the effluxpump inhibitor or fluoroquinolone.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard-pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal for 50% of thepopulation) and the ED50 (the does therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio betweenLD50/ED50. Compounds which exhibit large therapeutic indices arepreferred. The data obtained from these cell culture assays and animalstudies can be used in formulating a range of dosage for use in humansubjects. The dosage of such compounds lies preferably within a range ofcirculating concentrations that include the ED50 with little or notoxicity. The dosage may vary within this range depending upon thedosage form employed and the route of administration utilized. Forexample, in one embodiment the therapeutic serum concentration of anefflux pump inhibitor is in the range of 0.1–100 ug/ml.

VI. Microbes

Numerous different microbes are suitable for use in testing forcompounds that affect fluoroquinolone resistance or as sources ofmaterials for use in the instant assays or as targets for growthinhibition. The term “microbe” includes any microorganism having a anAcrAB-like efflux pump. Preferably unicellular microbes includingbacteria, fungi, or protozoa. In another embodiment, microbes suitablefor use in the invention are multicellular, e.g., parasites or fungi. Inpreferred embodiments, microbes are pathogenic for humans, animals, orplants. As such, any of these disclosed microbes may be used as intactcells or as sources of materials for cell-free assays as describedherein.

In preferred embodiments, microbes for use in the claimed methods arebacteria, either Gram-negative or Gram-positive bacteria. In a preferredembodiment, any bacteria that are shown to become resistant toantibiotics, e.g., to display MDR are appropriate for use in the claimedmethods.

In preferred embodiments, microbes suitable for testing are bacteriafrom the family Enterobacteriaceae. In more preferred embodimentsbacteria of a genus selected from the group consisting of: Escherichia,Proteus, Salmonella, Klebsiella, Providencia, Enterobacter,Burkholderia, Pseudomonas, Acinetobacter, Aeromonas, Haemophilus,Yersinia, Neisseria, and Erwinia, Rhodopseudomonas, or Burkholderia isused in the claimed assays.

In yet other embodiments, the microbes to be tested are Gram-positivebacteria and are from a genus selected from the group consisting of:Lactobacillus, Azorhizobium, Streptococcus, Pediococcus, Photobacterium,Bacillus, Enterococcus, Staphylococcus, Clostridium, Butyrivibrio,Sphingomonas, Rhodococcus, or Streptomyces

In yet other embodiments, the microbes to be tested are acid fastbacilli, e.g., from the genus Mycobacterium.

In still other embodiments, the microbes to be tested are, e.g.,selected from a genus selected from the group consisting of:Methanobacterium, Sulfolobus, Archaeoglobu, Rhodobacter, orSinorhizobium.

In other embodiments, the microbes to be tested are fungi. In apreferred embodiment the fungus is from the genus Mucor or Candida,e.g., Mucor racemosus or Candida albicans.

In yet other embodiments, the microbes to be tested are protozoa. In apreferred embodiment the microbe is a malaria or cryptosporidiumparasite.

In one embodiment, the microbe is resistant to one or more drugs. In apreferred embodiment, the microbe is highly resistant to one or moredrugs. In one embodiment, the drug is an antibiotic. In a preferredembodiment, the drug is a fluoroquinolone. In another embodiment, thedrug is a non-antibiotic. In another embodiment, the drug is triclosan.In one embodiment, a microbe comprises a mutation in a gene which is atarget of the drug to which the microbe is resistant, e.g.,topoisomerase, gyrase, or fabI gene. In another embodiment, a microbecomprises a mutation in at least two of a topoisomerase, gyrase, or fabIgene.

VII. Test Compounds

Compounds for testing in the instant methods can be derived from avariety of different sources and can be known or can be novel. In oneembodiment, libraries of compounds are tested in the instant methods toidentify AcrAB-like efflux pump blocking agents. In another embodiment,known compounds are tested in the instant methods to identify AcrAB-likeefflux pump blocking agents. In a preferred embodiment, compounds amongthe list of compounds generally regarded as safe (GRAS) by theEnvironmental Protection Agency are tested in the instant methods. Inone embodiment, an AcrAB-like efflux pump inhibitor is itself asubstrate of the pump.

A recent trend in medicinal chemistry includes the production ofmixtures of compounds, referred to as libraries. While the use oflibraries of peptides is well established in the art, new techniqueshave been developed which have allowed the production of mixtures ofother compounds, such as benzodiazepines (Bunin et al. 1992. J. Am.Chem. Soc. 114:10987; DeWitt et al. 1993. Proc. Natl. Acad. Sci. USA90:6909) peptoids (Zuckermann. 1994. J. Med. Chem. 37:2678)oligocarbamates (Cho et al. 1993. Science. 261:1303), and hydantoins(DeWitt et al. supra). Rebek et al. have described an approach for thesynthesis of molecular libraries of small organic molecules with adiversity of 104–105 (Carell et al. 1994. Angew. Chem. Int. Ed. Engl.33:2059; Carell et al. Angew. Chem. Int. Ed. Engl. 1994. 33:2061).

The compounds for screening in the assays of the present invention canbe obtained using any of the numerous approaches in combinatoriallibrary methods known in the art, including: biological libraries;spatially addressable parallel solid phase or solution phase libraries,synthetic library methods requiring deconvolution, the ‘one-beadone-compound’ library method, and synthetic library methods usingaffinity chromatography selection. The biological library approach islimited to peptide libraries, while the other four approaches areapplicable to peptide, non-peptide oligomer or small molecule librariesof compounds (Lam, K. S. Anticancer Drug Des. 1997. 12:145).

Exemplary compounds which can be screened for activity include, but arenot limited to, peptides, nucleic acids, carbohydrates, small organicmolecules (e.g., polyketides) (Cane et al. 1998. Science 282:63), andnatural product extract libraries. In one embodiment, the test compoundis a peptide or peptidomimetic. In another, preferred embodiment, thecompounds are small, organic non-peptidic compounds.

Other exemplary methods for the synthesis of molecular libraries can befound in the art, for example in: Erb et al. 1994. Proc. Natl. Acad.Sci. USA 91:11422; Horwell et al. 1996 Immunopharmacology 33:68; and inGallop et al. 1994. J. Med. Chem. 37:1233.

Libraries of compounds may be presented in solution (e.g., Houghten(1992) Biotechniques 13:412–421), or on beads (Lam (1991) Nature354:82–84), chips (Fodor (1993) Nature 364:555–556), bacteria (LadnerU.S. Pat. No. 5,223,409), spores (Ladner U.S. Pat. No. '409), plasmids(Cull et al. (1992) Proc Natl Acad Sci USA 89:1865–1869) or on phage(Scott and Smith (1990) Science 249:386–390); (Devlin (1990) Science249:404–406); (Cwirla et al. (1990) Proc. Natl. Acad. Sci.87:6378–6382); (Felici (1991) J. Mol. Biol. 222:301–310); (Ladnersupra). Other types of peptide libraries may also be expressed, see, forexample, U.S. Pat. Nos. 5,270,181 and 5,292,646). In still anotherembodiment, combinatorial polypeptides can be produced from a cDNAlibrary.

VIII. Pharmaceutical Compositions

The invention provides pharmaceutically acceptable compositions whichinclude a therapeutically-effective amount or dose of an efflux pumpinhibitor and one or more pharmaceutically acceptable carriers(additives) and/or diluents. A composition can also include a secondantimicrobial agent, e.g., an antimicrobial compound, preferably anantibiotic, e.g., a fluoroquinolone.

As described in detail below, the pharmaceutical compositions can beformulated for administration in solid or liquid form, including thoseadapted for the following: (1) oral administration, for example,drenches (aqueous or non-aqueous solutions or suspensions), tablets,boluses, powders, granules, pastes; (2) parenteral administration, forexample, by subcutaneous, intramuscular or intravenous injection as, forexample, a sterile solution or suspension; (3) topical application, forexample, as a cream, ointment or spray applied to the skin; (4)intravaginally or intrarectally, for example, as a pessary, cream, foam,or suppository; or (5) aerosol, for example, as an aqueous aerosol,liposomal preparation or solid particles containing the compound.

The phrase “pharmaceutically-acceptable carrier” as used herein means apharmaceutically-acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, solvent or encapsulatingmaterial, involved in carrying or transporting the antimicrobial agentsor compounds of the invention from one organ, or portion of the body, toanother organ, or portion of the body without affecting its biologicaleffect. Each carrier should be “acceptable” in the sense of beingcompatible with the other ingredients of the composition and notinjurious to the subject. Some examples of materials which can serve aspharmaceutically-acceptable carriers include: (1) sugars, such aslactose, glucose and sucrose; (2) starches, such as corn starch andpotato starch; (3) cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4)powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients,such as cocoa butter and suppository waxes; (9) oils, such as peanutoil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters,such as ethyl oleate and ethyl laurate; (13) agar; (14) bufferingagents, such as magnesium hydroxide and aluminum hydroxide; (15) alginicacid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer'ssolution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21)other non-toxic compatible substances employed in pharmaceuticalcompositions. Proper fluidity can be maintained, for example, by the useof coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain additional agents, such aspreservatives, wetting agents, emulsifying agents and dispersing agents.Prevention of the action of microorganisms may be ensured by theinclusion of various antibacterial and antifungal agents, for example,paraben, chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include isotonic agents, such as sugars, sodium chloride,and the like into the compositions. In addition, prolonged absorption ofthe injectable pharmaceutical form may be brought about by the inclusionof agents which delay absorption such as aluminum monostearate andgelatin.

In some cases, in order to prolong the effect of a drug, it is desirableto slow the absorption of the drug from subcutaneous or intramuscularinjection. This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material having poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolutionwhich, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally-administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle.

Pharmaceutical compositions of the present invention may be administeredto epithelial surfaces of the body orally, parenterally, topically,rectally, nasally, intravaginally, intracisternally. They are of coursegiven by forms suitable for each administration route. For example, theyare administered in tablets or capsule form, by injection, inhalation,eye lotion, ointment, etc., administration by injection, infusion orinhalation; topical by lotion or ointment; and rectal or vaginalsuppositories.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal and intrasternal injection and infusion.

The phrases “systemic administration,” “administered systemically,”“peripheral administration” and “administered peripherally” as usedherein mean the administration of a sucrose octasulfate and/or anantibacterial or a contraceptive agent, drug or other material otherthan directly into the central nervous system, such that it enters thesubject's system and, thus, is subject to metabolism and other likeprocesses, for example, subcutaneous administration.

In some methods, the compositions of the invention can be topicallyadministered to any epithelial surface. An “epithelial surface”according to this invention is defined as an area of tissue that coversexternal surfaces of a body, or which and lines hollow structuresincluding, but not limited to, cutaneous and mucosal surfaces. Suchepithelial surfaces include oral, pharyngeal, esophageal, pulmonary,ocular, aural, nasal, buccal, lingual, vaginal, cervical, genitourinary,alimentary, and anorectal surfaces.

Compositions can be formulated in a variety of conventional formsemployed for topical administration. These include, for example,semi-solid and liquid dosage forms, such as liquid solutions orsuspensions, suppositories, douches, enemas, gels, creams, emulsions,lotions, slurries, powders, sprays, lipsticks, foams, pastes,toothpastes, ointments, salves, balms, douches, drops, troches, chewinggums, lozenges, mouthwashes rinses.

Conventionally used carriers for topical applications include pectin,gelatin and derivatives thereof, polylactic acid or polyglycolic acidpolymers or copolymers thereof, cellulose derivatives such as methylcellulose, carboxymethyl cellulose, or oxidized cellulose, guar gum,acacia gum, karaya gum, tragacanth gum, bentonite, agar, carbomer,bladderwrack, ceratonia, dextran and derivatives thereof, ghatti gum,hectorite, ispaghula husk, polyvinypyrrolidone, silica and derivativesthereof, xanthan gum, kaolin, talc, starch and derivatives thereof,paraffin, water, vegetable and animal oils, polyethylene, polyethyleneoxide, polyethylene glycol, polypropylene glycol, glycerol, ethanol,propanol, propylene glycol (glycols, alcohols), fixed oils, sodium,potassium, aluminum, magnesium or calcium salts (such as chloride,carbonate, bicarbonate, citrate, gluconate, lactate, acetate, gluceptateor tartrate).

Such compositions can be particularly useful, for example, for treatmentor prevention of an unwanted cell, e.g., vaginal Neisseria gonorrhea, orinfections of the oral cavity, including cold sores, infections of eye,the skin, or the lower intestinal tract. Standard composition strategiesfor topical agents can be applied to the antimicrobial compounds, orpharmaceutically acceptable salts thereof in order to enhance thepersistence and residence time of the drug, and to improve theprophylactic efficacy achieved.

For topical application to be used in the lower intestinal tract orvaginally, a rectal suppository, a suitable enema, a gel, an ointment, asolution, a suspension or an insert can be used. Topical transdermalpatches may also be used. Transdermal patches have the added advantageof providing controlled delivery of the compositions of the invention tothe body. Such dosage forms can be made by dissolving or dispersing theagent in the proper medium.

Compositions of the invention can be administered in the form ofsuppositories for rectal or vaginal administration. These can beprepared by mixing the agent with a suitable non-irritating carrierwhich is solid at room temperature but liquid at rectal temperature andtherefore will melt in the rectum or vagina to release the drug. Suchmaterials include cocoa butter, beeswax, polyethylene glycols, asuppository wax or a salicylate, and which is solid at room temperature,but liquid at body temperature and, therefore, will melt in the rectumor vaginal cavity and release the active agent.

Compositions which are suitable for vaginal administration also includepessaries, tampons, creams, gels, pastes, foams, films, or spraycompositions containing such carriers as are known in the art to beappropriate. The carrier employed in the sucroseoctasulfate/contraceptive agent should be compatible with vaginaladministration and/or coating of contraceptive devices. Combinations canbe in solid, semi-solid and liquid dosage forms; such as diaphragm,jelly, douches, foams, films, ointments, creams, balms, gels, salves,pastes, slurries, vaginal suppositories, sexual lubricants, and coatingsfor devices, such as condoms, contraceptive sponges, cervical caps anddiaphragms.

For ophthalmic applications, the pharmaceutical compositions can beformulated as micronized suspensions in isotonic, pH adjusted sterilesaline, or, preferably, as solutions in isotonic, pH adjusted sterilesaline, either with or without a preservative such as benzylalkoniumchloride. Alternatively, for ophthalmic uses, the compositions can beformulated in an ointment such as petrolium. Exemplary ophthalmiccompositions include eye ointments, powders, solutions and the like.

Powders and sprays can contain, in addition to sucrose octasulfateand/or antibiotic or contraceptive agent(s), carriers such as lactose,talc, silicic acid, aluminum hydroxide, calcium silicates and polyamidepowder, or mixtures of these substances. Sprays can additionally containcustomary propellants, such as chlorofluorohydrocarbons and volatileunsubstituted hydrocarbons, such as butane and propane.

Ordinarily, an aqueous aerosol is made by formulating an aqueoussolution or suspension of the agent together with conventionalpharmaceutically acceptable carriers and stabilizers. The carriers andstabilizers vary with the requirements of the particular compound, buttypically include nonionic surfactants (Tweens, Pluronics, orpolyethylene glycol), innocuous proteins like serum albumin, sorbitanesters, oleic acid, lecithin, amino acids such as glycine, buffers,salts, sugars or sugar alcohols. Aerosols generally are prepared fromisotonic solutions.

Compositions of the invention can also be orally administered in anyorally-acceptable dosage form including, but not limited to, capsules,cachets, pills, tablets, lozenges (using a flavored basis, usuallysucrose and acacia or tragacanth), powders, granules, or as a solutionor a suspension in an aqueous or non-aqueous liquid, or as anoil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup,or as pastilles (using an inert base, such as gelatin and glycerin, orsucrose and acacia) and/or as mouth washes and the like, each containinga predetermined amount of sucrose octasulfate and/or antibiotic orcontraceptive agent(s) as an active ingredient. A compound may also beadministered as a bolus, electuary or paste. In the case of tablets fororal use, carriers which are commonly used include lactose and cornstarch. Lubricating agents, such as magnesium stearate, are alsotypically added. For oral administration in a capsule form, usefuldiluents include lactose and dried corn starch. When aqueous suspensionsare required for oral use, the active ingredient is combined withemulsifying and suspending agents. If desired, certain sweetening,flavoring or coloring agents may also be added.

Tablets, and other solid dosage forms, such as dragees, capsules, pillsand granules, may be scored or prepared with coatings and shells, suchas enteric coatings and other coatings well known in thepharmaceutical-formulating art. They may also be formulated so as toprovide slow or controlled release of the active ingredient thereinusing, for example, hydroxypropylmethyl cellulose in varying proportionsto provide the desired release profile, other polymer matrices,liposomes and/or microspheres. They may be sterilized by, for example,filtration through a bacteria-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved in sterile water, or some other sterile injectable mediumimmediately before use. These compositions may also optionally containopacifying agents and may be of a composition that they release theactive ingredient(s) only, or preferentially, in a certain portion ofthe gastrointestinal tract, optionally, in a delayed manner. Examples ofembedding compositions which can be used include polymeric substancesand waxes. The active ingredient can also be in micro-encapsulated form,if appropriate, with one or more of the above-described excipients.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, microemulsions, solutions, suspensions, syrups andelixirs. In addition to the active ingredient, the liquid dosage formsmay contain inert diluents commonly used in the art, such as, forexample, water or other solvents, solubilizing agents and emulsifiers,such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethylacetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyleneglycol, oils (in particular, cottonseed, groundnut, corn, germ, olive,castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethyleneglycols and fatty acid esters of sorbitan, and mixtures thereof.

Besides inert diluents, the oral compositions can also include adjuvantssuch as wetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming and preservative agents.

Suspensions, in addition to the antimicrobial agent(s) may containsuspending agents as, for example, ethoxylated isostearyl alcohols,polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth,and mixtures thereof.

Sterile injectable forms of the compositions of this invention can beaqueous or oleaginous suspensions. These suspensions may be formulatedaccording to techniques known in the art using suitable dispersing orwetting agents and suspending agents. Wetting agents, emulsifiers andlubricants, such as sodium lauryl sulfate and magnesium stearate, aswell as coloring agents, release agents, coating agents, sweetening,flavoring and perfuming agents, preservatives and antioxidants can alsobe present in the compositions.

The sterile injectable preparation may also be a sterile injectablesolution or suspension in a nontoxic parenterally-acceptable diluent orsolvent, for example as a solution in 1,3-butanediol. Among theacceptable vehicles and solvents that may be employed are water,Ringer's solution and isotonic sodium chloride solution. In addition,sterile, fixed oils are conventionally employed as a solvent orsuspending medium. For this purpose, any bland fixed oil may be employedincluding synthetic mono- or di-glycerides. Fatty acids, such as oleicacid and its glyceride derivatives are useful in the preparation ofinjectables, as are natural pharmaceutically-acceptable oils, such asolive oil or castor oil, especially in their polyoxyethylated versions.These oil solutions or suspensions may also contain a long-chain alcoholdiluent or dispersant.

In the case of AcrAB efflux pump inhibitors which are antisense nucleicacid molecules, the optimal course of administration of the oligomersmay vary depending upon the desired result or on the subject to betreated. As used in this context “administration” refers to contactingcells with oligomers. The dosage of antisense molecule may be adjustedto optimally reduce expression of a protein translated from a targetmRNA, e.g., as measured by a readout of RNA stability or by atherapeutic response, without undue experimentation. For example,expression of the protein encoded by the nucleic acid target can bemeasured to determine whether or dosage regimen needs to be adjustedaccordingly. In addition, an increase or decrease in RNA and/or proteinlevels in a cell or produced by a cell can be measured using any artrecognized technique. By determining whether transcription has beendecreased, the effectiveness of the antisense molecule in inducing thecleavage of the target RNA can be determined.

As used herein, “pharmaceutically acceptable carrier” includesappropriate solvents, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents, and thelike. The use of such media and agents for pharmaceutical activesubstances is well known in the art. Except insofar as any conventionalmedia or agent is incompatible with the active ingredient, it can beused in the therapeutic compositions. Supplementary active ingredientscan also be incorporated into the compositions.

Antisense molecule may be incorporated into liposomes or liposomesmodified with polyethylene glycol or admixed with cationic lipids forparenteral administration. Incorporation of additional substances intothe liposome, for example, antibodies reactive against membrane proteinsfound on specific target microbes, can help target the antisensemolecule to specific cell types.

Moreover, the present invention provides for administering the subjectoligomers with an osmotic pump providing continuous infusion of suchantisense molecules, for example, as described in Rataiczak et at. (1992Proc. Natl. Acad. Sci. USA 89:11823–11827). Such osmotic pumps arecommercially available, e.g., from Alzet Inc. (Palo Alto, Calif.).Topical administration and parenteral administration in a cationic lipidcarrier are preferred.

With respect to in vivo applications, the formulations of the presentinvention can be administered to a patient in a variety of forms adaptedto the chosen route of administration, namely, parenterally, orally, orintraperitoneally. Parenteral administration, which is preferred,includes administration by the following routes: intravenous;intramuscular; interstitially; intraarterially; subcutaneous; intraocular; intrasynovial; trans epithelial, including transdermal;pulmonary via inhalation; ophthalmic; sublingual and buccal; topically,including ophthalmic; dermal; ocular; rectal; and nasal inhalation viainsufflation. Intravenous administration is preferred among the routesof parenteral administration.

Pharmaceutical preparations for parenteral administration includeaqueous solutions of the active compounds in water-soluble orwater-dispersible form. In addition, suspensions of the active compoundsas appropriate oily injection suspensions may be administered. Suitablelipophilic solvents or vehicles include fatty oils, for example, sesameoil, or synthetic fatty acid esters, for example, ethyl oleate ortriglycerides. Aqueous injection suspensions may contain substanceswhich increase the viscosity of the suspension include, for example,sodium carboxymethyl cellulose, sorbitol, and/or dextran, optionally,the suspension may also contain stabilizers.

Drug delivery vehicles can be chosen e.g., for in vitro, for systemic,or for topical administration. These vehicles can be designed to serveas a slow release reservoir or to deliver their contents directly to thetarget cell. An advantage of using some direct delivery drug vehicles isthat multiple molecules are delivered per uptake. Such vehicles havebeen shown to increase the circulation half-life of drugs that wouldotherwise be rapidly cleared from the blood stream. Some examples ofsuch specialized drug delivery vehicles which fall into this categoryare liposomes, hydrogels, cyclodextrins, biodegradable nanocapsules, andbioadhesive microspheres.

The described antisense molecules may be administered systemically to asubject. Systemic absorption refers to the entry of drugs into the bloodstream followed by distribution throughout the entire body.Administration routes which lead to systemic absorption include:intravenous, subcutaneous, intraperitoneal, and intranasal. Each ofthese administration routes delivers the antisense molecule toaccessible diseased cells. Following subcutaneous administration, thetherapeutic agent drains into local lymph nodes and proceeds through thelymphatic network into the circulation. The rate of entry into thecirculation has been shown to be a function of molecular weight or size.The use of a liposome or other drug carrier localizes the antisensemolecule at the lymph node. The antisense molecule can be modified todiffuse into the cell, or the liposome can directly participate in thedelivery of either the unmodified or modified oligomer into the cell.

For prophylactic applications, the pharmaceutical composition of theinvention can be applied prior to physical contact with a microbe. Thetiming of application prior to physical contact can be optimized tomaximize the prophylactic effectiveness of the compound. The timing ofapplication will vary depending on the mode of administration, theepithelial surface to which it is applied, the surface area, doses, thestability and effectiveness of composition under the pH of theepithelial surface, the frequency of application, e.g., singleapplication or multiple applications. Preferably, the timing ofapplication can be determined such that a single application ofcomposition is sufficient. One skilled in the art will be able todetermine the most appropriate time interval required to maximizeprophylactic effectiveness of the compound.

One of ordinary skill in the art can determine and prescribe theeffective amount of the pharmaceutical composition required. Forexample, one could start doses at levels lower than that required inorder to achieve the desired therapeutic effect and gradually increasethe dosage until the desired effect is achieved. In general, a suitabledaily dose of a composition of the invention will be that amount of thecomposition which is the lowest dose effective to produce a therapeuticeffect. Such an effective dose will generally depend upon the factorsdescribed above. It is preferred that administration be intravenous,intracoronary, intramuscular, intraperitoneal, or subcutaneous.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of cell biology, cell culture,molecular biology, genetics, microbiology, recombinant DNA, andimmunology, which are within the skill of the art. Such techniques areexplained fully in the literature. See, for example, Genetics; MolecularCloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, J. et al. (ColdSpring Harbor Laboratory Press (1989)); Short Protocols in MolecularBiology, 3rd Ed., ed. by Ausubel, F. et al. (Wiley, NY (1995)); DNACloning, Volumes I and II (D. N. Glover ed., 1985); OligonucleotideSynthesis (M. J. Gait ed. (1984)); Mullis et al. U.S. Pat. No.4,683,195; Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds.(1984)); the treatise, Methods In Enzymology (Academic Press, Inc.,N.Y); Immunochemical Methods In Cell And Molecular Biology (Mayer andWalker, eds., Academic Press, London (1987)); Handbook Of ExperimentalImmunology, Volumes I–IV (D. M. Weir and C. C. Blackwell, eds. (1986));and Miller, J. Experiments in Molecular Genetics (Cold Spring HarborPress, Cold Spring Harbor, N.Y. (1972)).

The contents of all references, pending patent applications andpublished patents, cited throughout this application are herebyexpressly incorporated by reference.

The invention is further illustrated by the following examples, whichshould not be construed as further limiting.

EXAMPLES Example 1 Preventing Efflux Via an AcrAB Pump Renders HighlyResistant Microbes Comprising Chromosomal Mutations in Drug Target GenesSensitive to Drugs

Ineffectiveness of Gyrase Mutations in Escherichia coli in the Absenceof the AcrAB Multidrug Efflux Pump.

Fluoroquinolones [FQs] inhibit growth of wild-type Escherichia coli atvery low concentrations. While FQ efflux is present in wild-type E. colicells, its importance is minimal except when enhanced during thedevelopment of clinical resistance. To understand the role of the AcrABefflux pump, which is under control of the mar and sox regulons, on thelevel of susceptibility and clinical resistance to FQs in E. coli, twoseries of E coli K-12 cells (derived from wild type AG100 and from theisogenic Mar mutant AG102) with FQ resistance were generated byamplification on ofloxacin [ofx]-containing plates in-vitro andmutations conferring resistance were characterized. P1-transduction wasused to knock out AcrAB. Energy-dependent uptake of radiolabelledciprofloxacin [cfx] into whole cells was measured in the presence andabsence of AcrAB, and ofx MICs were determined by broth microdilution.In AG100, ofx resistance could be enhanced from 0.03 mg/l to 4 mg/l(128-fold) in four steps which included (in the order of occurrence): agyrA mutation, a mar mutation, an undefined mutation, and a 2^(nd) gyrAmutation. AG102, starting out as a Mar mutant, was amplified in threesteps from 0.125 mg/l to 8 mg/l (64-fold), which included one gyrAmutation and two as yet undefined mutations. Knock-out of AcrAB reducedthe ofx MICs by factors of 4 through 128, producing a dramatic decreasein resistance mediated by topoisomerase mutations. Ofx MICs±AcrAB wereas follows (in mg/l): AG100 series: 0.03/<0.015, 0.25/0.06, 1.0/0.06,2.0/0.06, 4.0/0.125; AG102 series: 0.125/<0.015, 1.0/0.06, 4.0/0.06,8.0/0.06. Active efflux of cfx was seen in the presence of the AcrABefflux pump. Drug accumulation in energized, AcrAB-deleted mutants,however, was equivalent to that in de-energized cells, i.e., activeefflux was completely abolished by deletion of AcrAB. Thus, the AcrABmultidrug efflux pump is the major, if not only, efflux pump in E. coliwhich controls the intracellular concentration of cfx. In its absence,high intracellular drug concentrations render topoisomerase mutationsrelatively ineffective in achieving clinical FQ resistance in E. coli.

Deletion of the AcrAB Efflux Pump Greatly Reduces FluoroquinoloneResistance in Gyrase Mutants of Escherichia coli

Fluoroquinolone-resistant mutants were selected from a wild-typeEscherichia coli K12 strain and its Mar mutant by stepwise exposure toincreasing levels of ofloxacin on solid medium. Analysis of mutationalsteps by Northern (RNA) blot analysis, sequencing and accumulationstudies with radiolabelled ciprofloxacin showed that mutations in thetarget gene gyrA, the regulatory gene marR and additional, as yetunidentified genes probably affecting AcrAB-mediated efflux ofciprofloxacin all contributed to fluoroquinolone resistance.Inactivation of the acrAB locus made all strains hypersusceptible tofluoroquinolones and certain other unrelated drugs. These studiesindicate that wild-type function of the AcrAB efflux pump is requiredfor expression of clinical fluoroquinolone resistance mediated bytopoisomerase mutations.

Fluoroquinolone (FQ) resistance in Escherichia coli can be caused bymutations in the target proteins of the drugs, DNA gyrase (Hooper, D. C.et al., 1987. The American Journal of Medicine 82:12–20; Piddock, L. J.V. 1995. Drugs 49:29–35) and topoisomerase IV (Heisig, P. 1996.Antimicrob. Agents Chemother. 40:879–885). Mutations affectingregulatory genes such as marA (Cohen, S. P., et al. 1993. J. Bacteriol.175: 1484–1492, Cohen, S. P., et al. 1989. Antimicrob. Agents Chemother.33:1318–1325) orsoxS (Amábile-Cuevas, C. F. and B. Demple. 1991. NucleicAcids Research 19:4479–4484) also lead to resistance. The latter genesregulate intracellular drug concentrations, either by decreased uptakeand/or increased efflux of the drug (Alekshun, M. N. and S. B. Levy.1997. Agents Chemother. 41: 2067–2075). In E. coli, overexpression ofMarA causes decreased expression of the OmpF porin (Cohen, S. P., et al.1988. J. Bacteriol. 170:5416–5422) and increased AcrAB expression(Okusu, H., et al. 1996. J. Bacteriol. 178:306–308), thereby conferringresistance to a large number of antimicrobials (Alekshun, M. N. and S.B. Levy. 1997. Antimicrob. Agents Chemother. 41: 2067–2075)). Thesequence of events leading to high-level, clinically significant FQresistance is still poorly understood. Studies on clinical FQ-resistantstrains are of limited help because one usually cannot isolate theparental strain. Studies with mutants selected in vitro have beenlimited to descriptions of phenotypic differences (MICs, outer membraneprofiles, accumulation of fluoroquinolones) or focussed on mutations inthe regions of gyrA and/or parC which determine quinolone resistance(QRDR) (Heisig, P. 1996. Antimicrob. Agents Chemother. 40:879–885;Piddock, L. J. V., et al. 1991. J. Antimicrob. Chemother. 28:185–198;Tenney, J. H., et al. 1983. Antimicrob. Agents Chemother. 23:188–189;Watanabe, M., et al. 1990. Antimicrob. Agents Chemother. 34:173–175). Inthis in vitro study the role of the AcrAB efflux pump was studied in FQresistance mediated by topoisomerase mutations acquired during step-wiseselection on ofloxacin. The AcrAB efflux pump was found to be criticalto the FQ resistance level.

The following materials and methods were used in this example:

Antibiotics, chemicals and media. Ofloxacin (OFL) was kindly donated byHoechst, Frankfurt, Germany. Radiolabelled [¹⁴C-]ciprofloxacin was agenerous gift of the Bayer AG, Leverkusen, Germany. Carbonyl cyanidem-chlorophenylhydrazone (CCCP) was purchased from Sigma Chemical Co.,St. Louis, Mo., and organic solvents from Aldrich, Milwaukee, Wis.Strains were grown in LB broth (10 g of tryptone, 5 g of yeast extractand 10 g of NaCl per liter) unless otherwise noted.

Bacterial strains and plasmids. All strains were derivatives ofplasmid-free E. coli K-12 strain AG100 (George, A. M. and S. B. Levy.1983. J. Bacteriol. 155:531–540) and its Mar mutant AG112 which wasselected on tetracycline in two steps (this study). Wild-type E. coliGC4468, its derived Sox mutant JTG1078 (soxR105; (Greenberg, J. T., etal. 1991. J. Bact 173:4433–4439)) and plasmid pSXS bearing the soxS geneon a 432 bp-fragment (Amábile-Cuevas, C. F. and B. Demple. 1991. NucleicAcids Research 19:4479–4484). Strain JZM120 was as described (Ma, D., etal. 1995. Mol Microbiol. 16:45–55)

Selection of fluoroquinolone-resistant mutants. 1-AG100, 2°-AG100,3*-AG100, 4*-AG100 and 1′-AG112, 2′-AG112, 3′-AG112 were sequential stepmutants derived from AG100 and AG112, respectively, on solid media. Foreach step, about 10¹¹ cells of an overnight culture were plated onseveral LB agar plates supplemented with increasing concentrations ofOFL. Single colonies were further purified on OFL-supplemented agarplates. Mutant 2°-AG100 came out of one series, while mutants 3*- and4*-AG100 came from a second series.

Susceptibility testing. MICs of selected antimicrobial agents weredetermined by a standard broth microdilution procedure withcation-adjusted Mueller-Hinton broth (Becton Dickinson, Cockeysville,Md.) and an inoculum of 5×10⁵ CFU/ml according to NCCLS performance andinterpretive guidelines (NCCLS. 1997. Methods for dilution antimicrobialsusceptibility. Tests for bacteria that grow aerobically—Fourth Edition;Approved Standard. NCCLS document M7-A4 (ISBN 1-56238-309-4). NCCLS, 940West Valley Road, Suite 1400, Wayne, Pa. 19087). Antimicrobial agents onthe commercially available microtiter plates (Merlin Diagnostics GmbH,Bornheim, Germany) included the FQs ciprofloxacin (CIP), enoxacin,fleroxacin, nor-flo-xacin, ofloxacin (OFL), pefloxacin, sparfloxacin(SPX), and trovafloxacin (TVA). They also included tetracycline (TET),chloramphenicol (CML), trimethoprim (TMP), cefoxitin (CFOX), cefaclor,cefixim and loracarbef. MICs of bile salts were determined by a brothmacrodilution procedure: sodium cholate and sodium deoxycholate wereserially diluted twofold in LB broth and tubes inoculated at 5×10⁵CFU/ml. MICs of both antibiotics and bile salts were determined twice inindependent experiments with reproducible results.

P1 transduction. AcrAB-deleted strains were constructed by P1transduction (Provence, D. L. and R. Curtiss III. 1994. In: P. Gerhardt,R. G. E. Murray, W. A. Wood, and N. R. Krieg. (ed.), Methods for Generaland Molecular Bacteriology. American Society for Microbiology,Washington. pp. 317–347) of acrAB::Tn903kan^(r) from strain JZM120 (Ma,D., et al. 1995. Mol Microbiol. 16:45–55) into AG100, AG112 and allderived FQ-resistant mutants. The AcrAB-deleted strains were designatedAG100AK, 1-through 4*-AG100AK, AG112AK and 1′- through 3′-AG112AK. Twoindependent transductants were saved for each recipient. Deletion ofAcrAB was confirmed by the absence of intact target DNA in a PCR assayand by greatly increased susceptibility to bile salts as previouslyreported (Thanassi, D. G., et al. 1997. J. Bacteriol. 179:2512–2518).

DNA sequencing. The QRDRs of gyrA (nucleotides 123 to 366) or parC(nucleotides 145 to 492) in the fluoroquinolone-resistant mutants wereamplified by PCR and purified by use of Qia-quick spin columns (Qia-gen,Hilden, Germany), as previously described (Conrad, S., M. et al. 1996.J. Antimicrob. Chemother. 38:443–455, Conrad, S., et al. 1996. Programand Abstracts of the 36th Interscience Conference of Anti-microbialAgents and Chemotherapy, New Orleans. Abstract C9:35). marOR wasamplified from bp 1311 to 1858 with primer pair ORAB2 and RK3 asdescribed earlier (Oethinger, M., et al. 1998. Agents Chemother.42:2089–2094). Direct cycle sequencing was performed in an automatic373A DNA Sequencer (Applied Biosystems).

RNA extraction and Northern blot analysis. Northern blot analysis wasperformed as previously described in detail (Oethinger, M., et al 1998.Antimicrob. Agents Chemother. 42:2089–2094). In brief, RNA was harvestedby a cesium chloride method from midlogarithmic phase cultures grown at30° C. For assessment of the state of the marRAB operon or the soxRSoperon, respectively, cultures were split and half the cultures inducedwith 5 mM sodium salicylate (marRAB operon) or 1.3 mM paraquat (soxRSoperon), respectively. The level of transcription from both operons wasassessed by hybridization of radiolabelled DNA probes (marA or soxS) tothe membrane-bound RNA (20 μg/lane), exposure on a PhosphoImager screen,and visualization with ImageQuant Software (Molecular Dynamics,Sunnyvale, Calif.), as described recently (Oethinger, M., et al. 1998.Antimicrob. Agents Chemother. 42:2089–2094).

Accumulation of [¹⁴C-]ciprofloxacin in whole cells. Cultures were grownto logarithmic phase in LB broth at 30° C., washed in 50 mM potassiumphosphate/0.2% glucose (pH 7.4), and resuspended in the same buffer toOD₆₀₀=5–7. [¹⁴C-]ciprofloxa-cin (specific activity: 59 mCi/mmol) wasadded to 10 μM. Accumulation was measured at equilibrium after 5 and 15min by dilution of 50 μl of cell-labeling suspension into 5 ml of 100 mMLiCl/50 mM KPO₄ (pH 7.4), collection of cells immediately on Gelmanmetricel mixed-cellulose ester membrane filters (pore size, 0.45 μm;Gelman Sciences Inc., Ann Arbor, Mich.), and washing with 5 ml of thesame buffer. Filters were dried, and radioactivity was assayed with aliquid scintillation counter, using Betafluor (National Diagnostics,Somerville, N.J.). Counting efficiency was 90%. Binding of radiolabel tofilters in the absence of cells was subtracted. For conversion purposes,10 μM CIP=3.15 μg/ml. When used, carbonyl cyanidem-chlorophenyl-hydrazone (CCCP), which destroys the proton motive force,was added to a final concentration of 200 μM, and accumulation ofciprofloxacin was assayed 5 and 15 min thereafter. The assay wasdesigned in a way to investigate up to five strains in one experimentand to include, in addition, AG112 as a control strain. Results werecalculated as accumulation of CIP in picomoles per OD₆₀₀ unit, where 1OD₆₀₀ unit represented the number of cells in 1 ml when the OD₆₀₀ wasequal to 1 (approximately 10⁹ E. coli cells, about 0.3 mg of protein).The ratio of CIP accumulation of energized cells divided by that ofde-energized (CCCP-treated) cells was used as an indirect measure ofactive efflux (Levy, S. B. 1992. Antimicrob. Agents Chemother.36:695–703). For these calculations, results obtained before (5 and 15min) and after adding CCCP (25 min and 35 min) were averaged and ratiosexpressed as % of de-energized (i.e. maximum) accumulation.

The mutation frequencies of the different step mutants ranged between8×10⁻⁸ and 10⁻¹⁰ which agrees well with previous data (Heisig, P. 1996.Antimicrob. Agents Chemother. 40:879–885, Piddock, L. J. V., et al.1991. J. Antimicrob. Chemother. 28:185–198, Watanabe, M., et al. 1990.Antimicrob. Agents Chemother. 34:173–175). None of the mutants wasdefective in growth. The first mutation step increased resistance to OFLby 8-fold in both AG100 and its Mar mutant AG112 (Table 1). Subsequentincreases were 2- to 4-fold in all steps (Table 1 and Table 2) yieldingthe highest MIC_(OFL) of 8 μg/ml in 3′-AG112. MICs of all FQs increasedin parallel, with the order of MICs being: OFL>CIP>TVA=SPX (Table 2).

Identification of chromosomal mutations in structural and regulatorygenes, and susceptibilities to unrelated antibiotics. Sequencing of theQRDRs of gyrA revealed that an identical point mutation at codon 87(substitution of glycine for aspartate) occurred during the firstmutation step in both AG100- and AG112-derived mutants (Table 1). Thesemutations led to an increase of MICs to FQs without an additionalmultiply resistance (Mar) phenotype. There are several reports about agyrA mutation being the first “visible” mutation in the chain of eventsto higher FQ resistance during stepwise in vitro mutagenesis (Heisig, P.1996. Antimicrob. Agents Chemother. 40:879–885, Kern, W. V., et al.1997. Program and Abstracts of the 37th Interscience Conference ofAntimicrobial Agents and Chemotherapy, Toronto (Abstract), Piddock, L.J. V., et al. 1991. J. Antimicrob. Chemother. 28:185–198). Theconcordance between AG100 and AG112 in the position of the first gyrAmutation may be coincidental.

The second step mutation in the AG100 background was a Mar mutation, asshown by overexpression of marRAB by Northern blot analysis of 2°-AG100.Such overexpression was found in three independently selected secondstep mutants of AG100. Constitutive overexpression of marA wasassociated with increased resistance to TET, CML and CFOX (Table 2).Retrospective sequencing of marOR showed that third step mutant 3*-AG100 had not been derived from 2°-AG100 (Table 1). Despite many efforts,the putative second step Mar mutant 2*-AG100 with a 1643G→T transitionin marOR could not be retrieved and further studied.

As expected (George, A. M. and S. B. Levy. 1983. J. Bacteriol.155:531–540), marRAB was derepressed in Mar mutant AG112 and allsubsequently derived mutants. Sequencing of marOR of the parental strainAG112 identified a 5 base pair deletion after the codon for amino acid12, resulting in deletion of one amino acid and change of the completeprotein sequence thereafter (Table 1). These data indicate that a singlemutation in the gyrA gene confers a somewhat higher resistance thanoverexpression of marA by itself (MIC_(OFL)=0.25 μg/ml (8-fold) vs.0.125 μg/ml (4-fold); 1-AG100 vs. AG112, Table 1) and that the twomutations are multiplicative (MIC_(OFL)=1 μg/ml (32-fold); 2°-AG100 and1′-AG112, Table 1). During further steps to higher FQ resistance onlyone mutant, 4*-AG100, acquired a second mutation in gyrA at codon 83,substituting leucine for serine (Table 1). No additional mutations ingyrA, parC or marOR could be identified in any of the more resistantmutants, nor did they constitutively overexpress soxS at any step.Mutant 2′-AG112, derived from gyrA/Mar double mutant 1′-AG112,dis-played increased resistance to multiple drugs (Table 2) with nofurther mutation in marOR. The additional mutation possibly leads toupregulation of acrAB. In contrast, next step mutant 3′-AG112 hadincreased resistance to only FQs and CFOX (Table 2). The molecular basisfor this resistance is also unknown.

Accumulation of CIP into whole energized cells reached a plateau by 5min and averaged 103 pmol CIP/ODU₆₀₀ in AG100. When the proton motiveforce was dissipated by adding 200 μM CCCP, accumulation of[¹⁴C-]ciprofloxacin doubled (201 pmol CIP/ODU₆₀₀). These findingsconfirmed earlier studies with norfloxacin (Cohen, S. P., et al. 1988.Antimicrob. Agents Chemother. 32:1187–1191) that FQ-susceptible E. colicells use energy to reduce FQ accumulation, i.e. show active efflux.This phenomenon was also observed for Proteus vulgaris using ofloxacin(Ishii, H., et al. 1991. J. Antimicrob. Chemother. 28:827–836). Incomparison, accumulation in AG112 was 54% of wild-type, averaging 56pmol CIP/ODU₆₀₀, and increased to 226 pmol CIP/ODU₆₀₀ after CCCP. Therapid accumulation of CIP or norfloxacin (Cohen, S. P., et al. 1988.Antimicrob. Agents Chemother. 32:1187–1191) to the level of the AG100parental strains upon deenergization of cells rules out down-regulationof outer membrane porins as the major mechanism of reduced drugaccumulation in Mar mutants. The amount of CIP accumulated by energizedfirst step mutants 1-AG100 and 1′-AG112 and the increase in drug uptakefollowing deenergization was virtually identical to that of parentalstrains AG100 and AG112, respectively. In second step Mar mutant2°-AG100, accumulation decreased to 65 pmol CIP/ODU₆₀₀ (63% ofwild-type; FIG. 1). Independently isolated mutants 3*- and 4*-AG100showed even greater reduced accumulation (16 pmol CIP/ODU₆₀₀=, 16% ofwild-type; and 20 pmol CIP/ODU₆₀₀, 19% of wild-type, respectively).Similarly, mutants 2′-AG112 and 3′-AG112 accumulated considerably lessCIP than Mar mutants AG112 and 1′-AG112 (26 pmol CIP/ODU₆₀₀ for bothstrains vs. 56 pmol CIP/ODU₆₀₀ and 57 pmol CIP/ODU₆₀₀, respectively, or25% vs. 54% and 55% of wild-type; FIG. 1). Due to more efficient effluxof the drug, the intracellular concentration of CIP in 3*-/4*-AG100 and2′-/3′-AG112 at high external drug levels is likely similar to that ofthe parental strains at lower levels, so that the mutants can survive ahigher extracellular FQ concentration. Whatever mechanism wasunderlying, it also increased the cells' resistance to TET, CML andCFOX.

Effects of deletion of acrAB. Upon deletion of the AcrAB multidrugefflux pump, active efflux of CIP was completely aborted in all strains;energized AG100 cells bearing the ΔacrAB deletion accumulated CIP tomore than twice the level seen for energized acrAB⁺ cells (FIG. 1). Nodifference in CIP uptake was noted between any strains bearing ΔacrABirrespective of their mutations in marRAB or gyrA (FIG. 1). Although allΔacrAB strains became profoundly hyper-susceptible to all FQs, mutantswith newly acquired mutations in gyrA (Table 1) probably still retainedthe expected fold FQ resistance (Table 2). This resistance, however, waswell below clinical significance. GyrA double mutant 4*-AG100ΔacrAB, forinstance, displayed a MIC_(OFL) of only 0.125 μg/ml. Interestingly, thedifferences in MICs for different FQs were no longer observed in ΔacrABstrains, e.g. in 2°-AG100 ΔacrAB⁺, MIC_(OFL)=MIC_(CIP)=MIC_(TVA)=0.06μg/ml, while in the acrAB⁺ strain 2°-AG100 MIC_(OFL)=1 μg/ml,MIC_(CIP)=0.5 μg/ml, MIC_(TVA)=0.25 μg/ml (Table 2). For SPX MICs of allΔacrAB mutants were below detection (MIC_(SPX) Δ0.015 μg/ml). This mayreflect a much higher proportion of drug being effluxed by the AcrABpump in energized cells. Deletion of acrAB also eliminated the multidrugresistance conferred by overexpression of marA, thus underliningprevious results that the AcrAB multidrug efflux pump plays a major rolein the antibiotic resistance phenotype of Mar mutants (Okusu, H., D. Ma,and H. Nikaido. 1996. J. Bacteriol. 178:306–308). Effects of additional,as yet unknown mutations were also completely abolished by deletion ofacrAB (Table 2). Thus, the effect of one or more additional mutation(s)on drug resistance may act via acrAB. Concluding Remarks. An earlierreport on selection of norfloxacin-resistant E. coli noted no change ofMICs of seven unrelated antibiotics during in vitro amplification ofFQ-resistant mutants (Tenney, J. H., et al. 1983. Antimicrob. AgentsChemother. 23:188–189). Another study showed that only two out of tensecond step mutants derived from a gyrA mutant displayed amultiply-resistant phenotype (Piddock, L. J. V., et al. 1991. J.Antimicrob. Chemother. 28:185–198).

The results of this study contrast with those previously reported: thestepwise sequence of mutational events during in vitro amplification ofFQ resistance in three independent series involved an initial gyrAmutation followed by a mutation causing overexpression of the mar locus.A marOR mutation was also seen during the second mutation step duringselection of FQ-resistant E. coli in vitro by others (Bagel, S., et al.1999. Antimicrob. Agents Chemother. 43:868–875).

In recent experiments, identical in vitro experimental conditions wereapplied to select mutants of two clinical E. coli isolates (Kern, W. V.,et al. 1997. Program and Abstracts of the 37th Interscience Conferenceof Antimicrobial Agents and Chemotherapy, Toronto (Abstract)). Since thesecond step mutant was a Mar mutant in one strain and a Sox mutant inthe other (Kern, W. V., et al. 1997. Program and Abstracts of the 37thInterscience Conference of Antimicrobial Agents and Chemotherapy,Toronto (Abstract)), thus, a mutation in a regulatory gene occursfrequently. However, studies on FQ-resistant E. coli of clinical originhave shown that the proportion of constitutive Mar or Sox mutants isonly between 10% and 15% (Oethinger, M., et al. 1998. Antimicrob. AgentsChemother. 42:2089–2094), a lower frequency than seen in the present invitro experiments.

In view of the high number of pumps in E. coli (Nikaido, H. 1996. J.Bacteriol. 178:5853–5859, Paulsen, I. T., et al. 1996. Microbiol. Rev.60:575–608) it is surprising that none of the other pumps activelyeffluxes CIP in the absence of AcrAB pump. Thus the AcrAB multidrugefflux pump appears to be the only or at least the most important pumpwhich uses CIP as substrate.

These findings correspond with recent data on triclosan susceptibilityof E. coli which was greatly affected by loss of the AcrAB pump(McMurry, L. M., et al. 1998. FEMS Microbiol. Lett. 166:305–309). Whileoverexpression of acrAB, marA, or soxS increased the cells resistance totriclosan about two-fold, a mutation in the target of triclosan, enoylreductase (encoded by fabI), rendered the cell about 100-fold moreresistant (McMurry, L. M., et al. 1998. Nature 394:531–532). However,deletion of acrAB reduced the resistance 10-fold in all strains,rendering the fabI mutation less effective, similar to the decrease ineffectiveness of topoisomerase mutations in the case of FQs.

The prominent finding of this work is that the AcrAB efflux pump has apowerful role in both the intrinsic and acquired level of resistance ofE. coli to FQs. These data show that efflux mechanisms decrease theaction of Fqs not only in Pseudomonas (Nikaido, H. 1996. J. Bacteriol178:5853–5859) but also in E. coli, even though in the latter organismthe drugs diffuse rapidly through the more permeable porin channels(Nikaido, H. 1996. J. Bacteriol. 178:5853–5859). Unidentified mutationsin chromosomal loci in addition to marOR or soxRS modulate the level ofresistance apparently by increasing efflux via AcrAB. Blockage of theAcrAB efflux pump would increase the potency of drugs such as FQs evenin the face of topoisomerase mutations.

TABLE 1 Fluoroquinolone Resistance Mutations in Escherichia coli AG100and its Mar mutant AG112 Fold CIP uptake Strain/ OFL MIC Increase ofSubstitution Mutation MarA (% of deenergized Mutant (μg/ml) OFL^(a) MICin gyrA In MarORAB Expression accumulation) AG100 0.03 — — — wild-type51 1-AG100 0.25 8 D87G None wild-type 43 2°-AG100 1 32 D87G Asp67Tyroverexpression 30 (1643G→T) 3*-AG100^(b) 2 64 D87G frameshift at aa12overexpression 9 (1485 + 1 bp) 4*-AG100^(b) 4 128 S83L, D87G frameshiftat aa12 overexpression 9 (1485 + 1 bp) AG112 0.125 4 — 5 bp deletionoverexpression 25 (Δ1481–1485) 1′-AG112 1 32 D87G 5 bp deletionoverexpression 24 (Δ1481–1485) 2′-AG112 4 128 D87G 5 bp deletionoverexpression 12 (Δ1481–1485) 3′-AG112 8 256 D87G 5 bp deletionoverexpression 9 (Δ1481–1485) ^(a)Ofloxacin; Fold change in MIC ascompared to AG100 (wild-type) ^(b)Mutants designated with an * were notderived from 2°-AG100 in * mutants.

TABLE 2 Effects of deletion of acrAB on susceptibility offluoroquinolone-resistant mutants of Escherichia coli AG100 and AG112MIC^(a) (μg/ml) OFL^(b) CIP^(b) TVA^(b) SPX^(b) TET^(b) CML^(b) CFOX^(b)Strain acrAB⁺ ΔacrAB acrAB⁺ ΔacrAB acrAB⁺ ΔacrAB acrAB⁺ ΔacrAB acrAB⁺ΔacrAB acrAB⁺ ΔacrAB acrAB⁺ ΔacrAB AG100 0.03 ≦0.015 ≦0.015 ≦0.0150.0625 ≦0.03 ≦0.015 ≦0.015 1 0.5 4 1 4 0.5 1-AG100 0.25 0.06 0.25 0.060.25 0.06 0.125 ≦0.015 2 1 4 1 4 0.5 2°-AG100 1 0.06 0.5 0.06 0.25 0.060.25 ≦0.015 2 0.5 16 1 16 0.5 3*-AG100 2 0.06 1 0.06 0.5 0.06 0.5 ≦0.0158 1 32 1 32 0.5 4*-AG100 4 0.125 2 0.125 2 0.125 2 ≦0.015 8 1 64 1 32 1AG112 0.125 ≦0.015 0.06 ≦0.015 0.125 ≦0.03 0.06 ≦0.015 4 1 16 1 32 11′-AG112 1 0.06 0.5 0.03 0.5 0.06 0.25 ≦0.015 4 0.5 32 1 16 1 2′-AG112 40.06 2 0.03 1 0.06 1 ≦0.015 16 1 64 1 32 0.5 3′-AG112 8 0.06 4 0.03 20.06 2 ≦0.015 8 0.5 64 1 64 0.5 ^(a)Determined as broth microdilutionaccording to NCCLS standards (#). Representative of experiments done induplicate. ^(b)OFL, ofloxacin; CIP, ciprofloxacin; TVA, trovafloxacin;SPX, sparfloxacin; TET, tetracycline; CML, chloramphenicol; CFOX,cefoxitin.

The contents of all references, pending patent applications andpublished patents, cited throughout this application are herebyexpressly incorporated by reference.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1. A method of screening for compounds which reduce antibioticresistance in a highly antibiotic resistant microbe comprising an AcrABor AcrAB-like efflux pump, wherein the microbe comprises at least two ofthe following traits: i) at least one chromosomal mutation in a geneencoding an antibiotic target that renders the microbe resistant to oneor more antibiotics; ii) a second mutation, to the same gene or adifferent gene than in (i), that further increases antibioticresistance, and iii) increased expression of at least one efflux pump,comprising: contacting the microbe with a test compound and measuringthe effect of the test compound on the activity of the AcrAB orAcrAB-like efflux pump, wherein compounds which inhibit the activity ofthe AcrAB or AcrAB-like efflux pump are identified as compounds whichreduce antibiotic resistance in the microbe.
 2. The method of claim 1,wherein the microbe comprises all three of the traits.
 3. The method ofclaim 1, wherein the microbe is highly resistant to fluoroquinolones. 4.The method of claim 1, wherein the at least one chromosomal mutation ispresent in a gene selected from the group consisting of: gyrase andtopoisomerase.
 5. The method of claim 1, wherein the microbe is a Gramnegative bacterium.
 6. The method of claim 1, wherein the microbefurther comprises functional porin channels.
 7. The method of claim 1,wherein the microbe is contacted with test compounds selected from alibrary of test compounds.
 8. The method of claim 1, wherein theactivity of the AcrAB or AcrAB-like efflux pump is determined bymeasuring efflux of an indicator compound which is a substrate of theefflux pump.
 9. The method of claim 1, wherein the activity of the AcrABor AcrAB-like efflux pump is determined by measuring growth of themicrobe in an antibiotic.
 10. The method of claim 1, wherein the effluxpump is AcrAB.
 11. A method of screening for compounds whichspecifically inhibit the activity of an AcrAB or AcrAB-like efflux pumpcomprising: i) contacting a microbe comprising an AcrAB or AcrAB-likeefflux and a non-AcrAB or non-AcrAB-like efflux pump and at least two ofthe following traits: i) at least one chromosomal mutation in a geneencoding a antibiotic target that renders the microbe resistant to oneor more antibiotics; ii) a second mutation, to the same gene or adifferent gene than in (i), that further increases antibioticresistance, and iii) increased expression of at least one efflux pump,with a test compound and an indicator compound; ii) testing the abilityof the test compound to inhibit the activity of the AcrAB or AcrAB-likeefflux pump; iii) testing the ability of the test compound to inhibitthe activity of the non-AcrAB or non-AcrAB efflux pump; iv) andidentifying compounds which inhibit the activity of the AcrAB orAcrAB-like efflux pump relative to the non-AcrAB or non-AcrAB-likeefflux pump to thereby identify compounds which specifically inhibit theactivity of the AcrAB or AcrAB-like efflux pump.
 12. The method of claim11, wherein the microbe is highly antibiotic resistant.
 13. The methodof claim 11, wherein the microbe is highly resistant tofluoroquinolones.
 14. The method of claim 11, wherein the at least onemutation is present in a gene selected from the group consisting of:gyrase and topoisomerase.
 15. The method of claim 11, wherein themicrobe is a Gram negative bacterium.
 16. The method of claim 11,wherein the microbe further comprises functional porin channels.
 17. Themethod of claim 11, wherein the microbe is contacted with test compoundsselected from a library of test compounds.
 18. The method of claim 11,wherein the activity of the AcrAB or AcrAB-like efflux pump isdetermined by measuring efflux of an indicator compound which is asubstrate of the efflux pump.
 19. The method of claim 11, wherein growthof the microbe in an antibiotic is measured.
 20. The method of claim 11,wherein the efflux pump is AcrAB.
 21. A method of treating an infectionin a subject caused by a microbe comprising an AcrAB or AcrAB-likeefflux pump and at least two of the following traits: i) at least onechromosomal mutation in a gene encoding a antibiotic target that rendersthe microbe resistant to one or more antibiotics; ii) a second mutation,to the same gene or a different gene than in (i), that further increasesantibiotic resistance, and iii) increased expression of at least oneefflux pump, comprising: contacting the microbe with an antibiotic towhich the microbe is resistant and an inhibitor of an acrAB oracrAB-like efflux pump such that the infection in the subject istreated.
 22. The method of claim 21, wherein the subject is treatedprophylactically.
 23. The method of claim 21, wherein the subject istreated therapeutically.
 24. The method of claim 21, wherein the microbeis highly resistant to fluoroquinolones.
 25. The method of claim 21,wherein the at least one mutation is present in a gene selected from thegroup consisting of: gyrase and topoisomerase.
 26. The method of claim21, wherein the microbe is a Gram negative bacterium.
 27. The method ofclaim 21, wherein the microbe further comprises functional porinchannels.